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Biomedical subjects

M Santoro

Publications and source records attributed to M Santoro.

At least 163 records · Page 9Linked to original sources

Developmental expression of the RET protooncogene.

The RET protooncogene encodes a transmembrane protein of the receptor-type tyrosine kinase family whose ligand has not yet been identified. Its activation in vivo is restricted to human carcinomas of the thyroid. In order to learn more about the possible role played by RET during normal development, we have examined its expression by performing in situ hybridization experiments on mouse embryos. Here, we show that the RET protooncogene is expressed during mouse embryogenesis in an unusual temporal and spatial manner. In fact, its expression was first detected around day 10 of gestation in the basal plate of the neural tube and in the developing encephalic ganglia, and later its pattern of expression was definitely established in neural structures, mostly in neural crest derivatives (spinal and encephalic ganglia). As far as the central nervous system is concerned, RET expression was confined to the ventral part of the midbrain from 12.5 days postcoitum (dpc) until birth. RET was also found to be expressed within structures of sensory organs such as the ganglial layer of the retina and the olfactory epithelium. A peculiar pattern of RET expression was clearly observed in the wall of the gut and in the nephrogenic zone of the developing kidney cortex, specifically in the metanephrogenic vesicles. Finally, RET was found to be expressed in the liver mostly between 12.5 dpc and 14.5 dpc. In conclusion, its expression in the early stages of embryogenesis suggests that RET may play a role in the differentiation of specific neural structures and the excretory system.

Animals↗

Cloning and characterization of H4 (D10S170), a gene involved in RET rearrangements in vivo.

H4(D10S170) is a gene which we isolated because of its frequent rearrangement with the RET proto-oncogene in vivo. Its fusion to RET generates the RET/PTC1 oncogene, which has been detected in about 20% of human thyroid papillary carcinomas. We have cloned and sequenced the cDNA corresponding to the H4(D10S170) gene from a human normal thyroid cDNA library. The nucleotide sequence of the H4(D10S170) 3 kb transcript shows no significant homology to known genes and contains an open reading frame (ORF) of 585 amino acids. H4(D10S170) predicted protein has no transmembrane domain and shows extensive regions in the alpha helical conformation, which are 30% homologous to the alpha-helical domains of several proteins including tropomyosin, vimentin, keratin and the tail region of myosin heavy chain. A putative SH3 binding site is present at the carboxy terminus, which suggests that H4(D10S170) might be a cytoskeletal protein.

Amino Acid Sequence↗

Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma.

The RET proto-oncogene encodes a transmembrane receptor of the tyrosine kinase family and has frequently been found activated in human thyroid carcinomas of the papillary subtype. In most cases the activation consisted of the fusion of its tyrosine-kinase domain with the 5'-terminal region of a gene designated H4 or D10S170. We have named the resulting H4/RET chimeric oncogene RET/PTC. Another activated form of the RET oncogene has subsequently been found in a thyroid carcinoma and is now referred to as RET/PTC2. Here we report the identification and cloning of a novel rearranged version of the RET oncogene in a human thyroid papillary carcinoma. In this case the tyrosine-kinase domain of RET was fused to a sequence 790 bp long belonging to a new gene that we have named RFG (RET Fused Gene). This novel chimeric oncogene has been designated RET/PTC3. In order to have more insights into the function of RFG we have completely cloned and sequenced its cDNA. RFG predicted amino-acid sequence does not have any significant homology to any already known genes and is ubiquitously expressed in human and mouse tissues. Finally we provide evidence indicating that the rearrangement leading to the generation of RET/PTC3 occurred in vivo in the original tumor DNA.

Amino Acid Sequence↗

Conduction abnormalities induced by sera of patients with multifocal motor neuropathy and anti-GM1 antibodies.

Increased titers of anti-GM1 antibodies have been associated with motor neuron disease and motor neuropathy with or without conduction block. To investigate the pathogenetic role of anti-GM1 antibodies we injected into rat tibial nerves sera from patients with multifocal motor neuropathy and conduction block (MMN) or progressive spinal muscular atrophy (PMA), both presenting anti-GM1 antibodies. Sera of patients with MMN produced reduction of amplitude and dispersion of compound muscle action potential from proximal stimulation. Morphometry revealed demyelination in 6.2% of fibers. Sera of patients with PMA did not produce clear-cut electrophysiological or morphological changes. Differential effects of sera from patients presenting high-titer anti-GM1 antibodies, but with distinct clinical syndromes, might depend on differences in anti-GM1 antibody affinity, valency, or ability to fix complement. Alternatively, circulating factors other than, or in addition to, anti-GM1 antibodies present in sera of patients with MMN, but not of PMA patients, might be responsible for conduction abnormalities and reproduce them after passive transfer.

Animals↗

Norepinephrine and thyrotropin stimulation of [Ca++]i in PC C13 a rat thyroid epithelial cell line: effect of transformation by E1A gene adenovirus and polyomavirus middle-T antigen gene.

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin.

Adenoviridae↗

Involvement of RET oncogene in human tumours: specificity of RET activation to thyroid tumours.

Non-thyroid neoplasia were analysed by Southern blot of genomic DNA and DNA prepared by reverse transcription and amplification by polymerase chain reaction (RT/PCR) for the activation of the RET oncogene. It is known that the rearrangement of RET occurs in about 10%-20% of human thyroid papillary carcinomas. None of 528 non-thyroid tumours showed rearrangement of the RET proto-oncogene, whereas three out of 30 thyroid papillary carcinomas were positive for RET activation. Therefore the activation of RET seems to be a somatic cell mutation specific to human thyroid carcinomas.

Base Sequence↗

Muscle rearrangement in patients with hemiparesis after stroke: an electrophysiological and morphological study.

Skeletal muscle changes were evaluated in patients suffering from hemiparesis after stroke. Concentric needle EMG and single fiber EMG of the paretic gastrocnemius medialis muscle were performed. Maximal amplitude of H, T and M responses in calf muscles of both the affected and the unaffected sides were determined by usual electrophysiological techniques. Muscle biopsy of the lateral gastrocnemius muscle of the affected side was performed to determine the distribution of fiber types and fiber sizes. Fibrillation activity and positive sharp waves occurred in paretic muscles in patients with more recent hemiparesis while the duration of motor unit potentials was prolonged in patients with long-lasting disease. The H/M ratio was increased on the paretic side. The percentage of type 1 fibers was augmented in most patients with normal mean diameter and low atrophy factor. The percentage of type 2 fibers was reduced with decreased mean diameters and with a high atrophy factor. Such changes may be related to inactivity or transsynaptic degeneration of type 2 motoneurons as a consequence of the interruption of the corticospinal tract. Increased percentage of type 1 fibers may be due to a collateral reinnervation process or a motor unit type transformation.

Aged↗

Inhibition of bacterial adhesion by sub-inhibitory concentrations: brodimoprim vs trimethoprim.

There is accumulating evidence that sub-inhibitory concentrations (sub-MICs) of many antibiotics are not without effect on bacteria and even though they do not kill bacteria, they are still able to interfere with some important aspects of bacterial cell function. The aim of the present study was to investigate the effect of sub-MICs of brodimoprim and trimethoprim on Escherichia coli and Staphylococcus aureus adhesiveness to human mucosal cells. Sub-MICs of brodimoprim down to 1/32 MIC (0.03 microgram/ml) significantly reduced the E. coli adhesion to human buccal epithelial cells and this inhibition was significantly higher than the corresponding pattern for trimethoprim. Adhesion of S. aureus was significantly reduced down to 1/16 MIC for both brodimoprim and trimethoprim but no significant differences resulted between the two patterns. 2,4 Diaminopyrimidines and related structures have a high affinity for the enzyme dihydrofolate reductase and this causes a reduction in the synthesis of essential purines, thus reducing also DNA and the synthesis or expression of surface adhesins.

Bacterial Adhesion↗

The Adenovirus E1A gene blocks the differentiation of a thyroid epithelial cell line, however the neoplastic phenotype is achieved only after cooperation with other oncogenes.

The PC Cl 3 cell line is a well characterized epithelial thyroid cell line of Fischer rat origin. This cell line has the peculiarity of retaining in vitro the typical markers of thyroid differentiation (i.e. thyroglobulin synthesis and secretion, iodide uptake and dependence on TSH for growth). The PC Cl 3 cells have been transfected with the E1A gene of Adenovirus 5. The E1A transfected cells, PC E1A, partially lost the dependency on TSH for growth and completely lost the ability to trap iodide and synthesize thyroglobulin; however they did not acquire the typical markers of the neoplastic phenotype. A highly malignant phenotype was achieved after infection of the PC E1A cells with retroviruses carrying the v-raf, v-abl and polyoma virus middle T oncogenes. In contrast, the PC E1A cells transfected with the E1B gene of Adenovirus were not tumorigenic at all, and those infected with retroviruses carrying oncogenes of the ras family displayed a very weak tumorigenic phenotype.

Adenovirus E1A Proteins↗

The TRK and RET tyrosine kinase oncogenes cooperate with ras in the neoplastic transformation of a rat thyroid epithelial cell line.

We have recently reported that about 50% of papillary thyroid carcinomas harbor an activated TRK or RET oncogene. Two retroviral vectors containing the activated TRK or RET/PTC oncogene have been used to infect a differentiated rat thyroid epithelial cell line, namely the PC Clone 3 cell line. Upon infection with the TRK virus, the PC Clone 3 cells lost only the ability to trap iodide and to express the thyroperoxidase gene. Conversely, when infected with the PTC virus, the PC Clone 3 cells completely lost all of their differentiated functions. However, both the PC-TRK and PC-PTC cell lines were unable to grow in soft agar, and they were not tumorigenic when injected into nude mice. A completely undifferentiated and malignant phenotype was obtained by the cooperation between the TRK or RET and the viral Ha-ras or Ki-ras oncogenes.

Animals↗

Characterization of an inversion on the long arm of chromosome 10 juxtaposing D10S170 and RET and creating the oncogenic sequence RET/PTC.

RET/PTC is a transforming sequence created by the fusion of the tyrosine kinase domain of the RET protooncogene with the 5' end of the locus D10S170 designated by probe H4 and is frequently found activated in human papillary thyroid carcinomas. RET and D10S170 have been mapped to contiguous regions of the long arm of chromosome 10: q11.2 and q21, respectively. To identify the mechanism leading to the generation of the oncogenic sequence RET/PTC, a combined cytogenetic and molecular analysis of several cases of papillary thyroid carcinomas was done. In four cases the results indicated that these tumors had RET/PTC activation and a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with breakpoints coincident with the regions where RET and D10S170 are located. Therefore, a chromosome 10q inversion provides the structural basis for the D10S170-RET fusion that forms the hybrid transforming sequence RET/PTC.

Blotting, Southern↗

Experimental conduction block induced by serum from a patient with anti-GM1 antibodies.

Increased titers of antibodies to GM1 ganglioside in humans are associated with lower motor neuron disease and predominantly motor neuropathy with or without conduction block. To investigate the possible mechanism of these antibodies, we injected the serum of a patient with anti-GM1 antibodies who had motor neuron disease and multifocal motor conduction block, into rat sciatic nerve. When injected with fresh human complement, the serum-induced conduction block with temporal dispersion and deposits of immunoglobulin were detected at the nodes of Ranvier. Electron microscopic studies revealed demyelination in 6.5% of the fibers. After preabsorption with GM1, the serum had no effect, suggesting that the anti-GM1 antibodies were responsible for the conduction abnormalities.

Animals↗

Patterns of reactivity of human anti-GM1 antibodies with spinal cord and motor neurons.

Human anti-GM1 antibodies from patients with lower motor neuron disease or predominantly motor neuropathy recognize carbohydrate determinants shared by GM1 and related glycolipids and glycoproteins, but the identity of the antigens to which they bind in tissue is unknown. We examined the binding of anti-GM1 antibodies with differing fine specificities to spinal cord, isolated motor neurons, and dorsal root ganglia neurons in order to characterize the tissue antigens. All anti-GM1 antibodies tested bound to the surface of bovine spinal motor neurons and immunostained the gray matter of unfixed sections of spinal cord. The staining was blocked by cholera toxin, which is specific for GM1, indicating that GM1 itself was the target antigen. Binding to white matter was more variable and depended on fixation and the fine specificities of the antibodies. The anti-GM1 antibodies did not bind to dorsal root ganglia neurons in tissue sections or in culture. These studies suggest that the autoantibodies might exert their effect, in part, by binding to GM1 on the surface of motor neurons, and that the absence of binding to dorsal root ganglia neurons might explain the lack of sensory abnormalities in affected patients.

Animals↗

Detection of RET oncogene activation in human papillary thyroid carcinomas by in situ hybridisation.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, named PTC, is a novel rearranged version of the ret proto-oncogene. In fact PTC is the product of the fusion of the tyrosine kinase domain of the ret proto-oncogene with the 5'-terminal region of another gene that we have named H4. The ret proto-oncogene shows a pattern of expression restricted to neuroendocrine tissue. Its fusion with H4 allows the expression of the activated form in thyroid papillary carcinomas. Therefore the detection of ret transcripts is a tool to investigate ret activation in thyroid neoplasms. Here we show the detection by in situ hybridisation, of activated ret transcripts in human thyroid papillary neoplasms that were positive for PTC activation by Southern blot analysis. We did not find any ret transcripts in papillary carcinomas negative for PTC activation, nor in normal thyroid and in non-papillary thyroid neoplasias.

3T3 Cells↗

A computer-assisted automatic method for myelinated nerve fiber morphometry.

This paper describes a computer-assisted automatic method developed for myelinated nerve fiber morphometry. IBAS image analysis system was used. One-micron thick sections of sural nerve were stained with p-phenylenediamine. Dark myelin sheaths, standing out against a pale background, were identified by a binary transformation of the computer-stored image. Different parameters of the fibers as well as of the axons can be measured. The automatic system allows operator-interactive manipulation, if required, to avoid inclusion of wrong histologic structures. A large spectrum of statistics facilities are available. We performed a comparison between automatic and semi-automatic analysis. Measurements resulted virtually overlapping, but time was three to four times shorter with automatic procedure. The method here described is accurate and reliable, and permits economy of time and effort.

Biopsy↗

Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, papillary thyroid carcinoma (PTC), is a novel rearranged version of the ret tyrosine-kinase protooncogene. Thyroid neoplasms include a broad spectrum of malignant tumors, ranging from well-differentiated tumors to undifferentiated anaplastic carcinomas. To determine the frequency of ret oncogene activation, we analyzed 286 cases of human thyroid tumors of diverse histologic types. We found the presence of an activated form of the ret oncogene in 33 (19%) of 177 papillary carcinomas. By contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic, and 18 medullary carcinomas, and 34 benign lesions, showed ret activation.

Base Sequence↗

Morphometric analysis of sciatic nerve and its main branches in the rabbit.

In the rabbit the myelinated fibres with the largest diameter originating from the sciatic nerve distribute mainly into the peroneal nerve, even though there is no difference in the density of myelinated fibres of the representative unit between the sciatic nerve and its branches. The distribution of the myelinated fibre diameters is unimodal in the sciatic nerve, but bimodal both in the peroneal and tibial nerves. The quotient axon diameter/fiber diameter (g ratio) shows a different trend between the sciatic nerve and the branches originating from it. Except for the peroneal nerve, the increase of the axon calibre corresponds to the reduction of the relative myelin sheath thickness and therefore to the reduction of internodal distance. The results show that the sciatic nerve and its terminal branches in the rabbit have morphometrical characteristics that are peculiar and differ from those of the other rodents.

Animals↗

Identification of the product of two oncogenic rearranged forms of the RET proto-oncogene in papillary thyroid carcinomas.

In papillary thyroid carcinomas, we have identified two tumor-specific rearrangements of the RET proto-oncogene leading to the formation of different transforming fusion products sharing the tyrosine kinase (tk) domain of the proto-oncogene and designated ptc-1 and ptc-2. We have analysed ptc-1 and ptc-2 products by immunoprecipitation with specific anti-RET antibodies followed by immunoblotting with the same reagent or with antibodies specific for phosphotyrosine (P-tyr) residues. The anti-RET antibodies were reactive with 64-kDa (p64ptc-1) and 81-kDa (p81ptc-2) proteins from lysates of ptc-1 and ptc-2 transformed cells, respectively, and identified two proteins of 140 kDa and 160 kDa from extracts of SK-N-SH, a neuroblastoma cell line previously shown to express two differently glycosylated forms of the normal RET product. The anti P-tyr antibodies, while detecting the same p64ptc-1 and p81ptc-2 proteins from ptc-1 and ptc-2 extracts, did not show any specific band in the neuroblastoma lysates. An additional set of experiments led us to conclude that, whereas the normal product of the RET proto-oncogene is a membrane-associated receptor-like molecule not intrinsically phosphorylated on tyrosine, both oncogenic forms of RET, ptc-1 and ptc-2, are constitutively phosphorylated on tyrosine, display an 'in vitro' autophosphorylation activity, are translocated from the membrane to the cytoplasm and are apparently unaffected by protein kinase C modulation.

3T3 Cells↗