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Biomedical subjects

A Villa

Publications and source records attributed to A Villa.

At least 217 records · Page 12Linked to original sources
PubMed1993

The endoplasmic reticulum-sarcoplasmic reticulum connection. II. Postnatal differentiation of the sarcoplasmic reticulum in skeletal muscle fibers.

The postnatal differentiation of sarcoplasmic reticulum (SR) of rabbit skeletal muscles (the slow-twitch soleus and the fast-twitch adductor muscles) was monitored between Days 1 and 12 by following on Western blots the expression and accumulation of molecular markers specific not only for the muscle endomembrane system, i.e., calsequestrin (CS) and the ryanodine-sensitive Ca2+ release channel, but also for the endoplasmic reticulum (ER) at large, i.e., BiP, calnexin (CN) and calreticulin. Our results demonstrate that SR development, documented by the increase of the SR fractional volume, terminal cisternae proliferation, and reorientation of triads, is accompanied by the accumulation of the SR-specific proteins and also of CN, with no change of the other ER general markers. Moreover, the distribution of two of the markers, BiP and CS, was investigated by immunocytochemistry at both the light and the electron microscope level. At Day 1 CS was found to be concentrated both within the few recognizable triad terminal cisternae and within the lumen of numerous, apparently discrete cisternae and tubules, widely scattered throughout both the contractile and the subplasmalemmal areas of the cytoplasm. These structures remain evident until Day 12, when most triad junctions have acquired proper configuration, composition and orientation. BiP, on the other hand, appears widely distributed within the ER/SR of the fibers. From the early stages of postnatal development it does colocalize with the Ca2+ binding protein in the lumen of the CS-rich structures and appears also within the longitudinal SR and the conventional ER cisternae.

Animals↗
PubMed1993

ZNF75: isolation of a cDNA clone of the KRAB zinc finger gene subfamily mapped in YACs 1 Mb telomeric of HPRT.

We have previously mapped a zinc finger genomic motif (ZNF75) to the Xq26 cytogenetic band by using a hybrid panel. Here, we report the isolation of the transcribed counterpart in a cDNA clone and its further localization. The cDNA clone, from a lung fibroblast library, is assembled from three exons, including a 289 amino acid (AA) long open reading frame containing a recently described motif, the Kruppel-associated box, 42 AA long, in exon 2. By comparison with other reported members of the subfamily, the exon-intron boundaries also appear to be very well conserved. Further analysis allowed us to map this gene 1 Mb downstream from the HPRT gene in the published YAC contig that extends across Xq26. Two other motifs, 87 and 78% homologous to ZNF75 at the amino acid level, were identified by PCR on total human DNA, but map outside Xq24-qter.

Amino Acid Sequence↗
PubMed1993

Tridimensional organization of Purkinje neuron cisternal stacks, a specialized endoplasmic reticulum subcompartment rich in inositol 1,4,5-trisphosphate receptors.

Stacks of regularly spaced, flat, smooth-surfaced endoplasmic reticulum cisternae frequently observed in both the cell body and dendrites of cerebellar Purkinje neurons, were previously shown by immunocytochemistry to be highly enriched in receptors for the second messenger, inositol 1,4,5-trisphosphate. Morphometric analyses have been carried out on randomly selected thin section images of rat Purkinje neurons to reveal the tridimensional organization of these structures. Individual stacked cisternae (on the average approximately 3.5 per stack) were shown to be separated from each other by a 23.5 nm space occupied by perpendicular bridges, approximately 20 nm in diameter, most probably composed by two apposed receptor homotetramer molecules, inserted into the parallel membranes in their hydrophobic domains. In the stacked membranes the density of the bridges was approximately 500 microns -2, corresponding to approximately 15% of the surface area. The lateral distribution of bridges was not random, but revealed regular distances that might correspond to unoccupied receptor slots. In each stack, the external cisternae were often in direct lumenal continuity with conventional elements of the endoplasmic reticulum, whereas the internal cisternae were not. Since continuities between stacked cisternae were never observed, the results indicate that the internal cisternae are at least transitorily discrete, i.e. they are not in permanent lumenal continuity with the rest of the endoplasmic reticulum. To our knowledge this is the first demonstration of a physical subcompartmentalization of the latter endomembrane system in a non-mitotic cells. A model for the biogenesis of cisternal stacks, based on the head-to-head binding and lateral interaction of the inositol 1,4,5-trisphosphate receptor molecules in the plane of the interacting membranes, is proposed and critically discussed.

Animals↗
PubMed1993

Multiple/heterogeneous Ca2+ stores in cerebellum Purkinje neurons.

1. The rapid and transient redistribution of Ca2+ from intracellular membrane-bound compartments (stores) is a key event of cell activation. 2. The cytological nature and molecular composition of such Ca2+ stores have been the object of intense investigation in recent years. 3. Here we review: (a) the current knowledge on intracellular Ca2+ stores of Purkinje neurons at the functional, biochemical, molecular, morphological and ultrastructural level; and discuss: (b) the relationship between Ca2+ stores and the endoplasmic reticulum, and (c) the occurrence of multiple/heterogeneous Ca2+ stores.

Animals↗
PubMed1993

Nicotinamide analogs and DNA-damaging agents deplete thyroid hormone receptor and c-erbA mRNA levels in pituitary GH1 cells.

Incubation of pituitary GH1 cells with N'-methylnicotinamide, nicotinamide and 3-acetylpyridine which inhibit nuclear ADP-ribosylation and/or the cellular concentration of its substrate NAD+ reduced the amount of nuclear thyroid hormone receptors in a time- and dose-dependent manner without altering the affinity of the receptors for the hormone. A transient activation of poly(ADP-ribose)polymerase by methyl methanesulfonate, ultraviolet irradiation or spermine caused a rapid depletion of cellular NAD+ content and was followed by a strong inhibition of ADP-ribosylation. These agents also produced a very rapid and marked reduction of receptor numbers. The decrease of receptors caused by the different compounds is not secondary to a generalized inhibition of protein synthesis or to an alteration in hormone availability. The abundance of c-erbA alpha and beta mRNAs, which encode thyroid hormone receptors, was reduced in cells treated with the compounds that decrease receptor number, thus suggesting that this effect is caused by a decrease in the expression of c-erbA genes.

Adenosine Diphosphate Ribose↗
PubMed1993

Preimplantation embryo sexing by polymerase chain reaction amplification of the sry gene on single mouse blastomeres.

Accurate and rapid sex determination of preimplantation embryos has great potential both in animal breeding and in human pathology. In the past, sex determination has been accomplished by cytogenetic or immunologic means and by polymerase chain reaction amplification of Y-chromosome-specific repetitive sequences. More recently, amplification of the Y-specific single-copy ZFY gene has been used in humans for sex determination of preimplantation embryos. The experiments reported here indicate that another Y-chromosome-specific single-copy gene, the sex-determining region gene (sry) can be successfully amplified from single mouse blastomeres. Blastocysts positive for sry amplification were reimplanted to foster mothers, and six of six newborns were male. We conclude that sry gene amplification can represent a good marker for embryo sex determination.

Animals↗
PubMed1993

The endoplasmic-sarcoplasmic reticulum of smooth muscle: immunocytochemistry of vas deferens fibers reveals specialized subcompartments differently equipped for the control of Ca2+ homeostasis.

Cryosection immunofluorescence and immunogold labeling with antibodies against specific markers were used in rat vas deferens smooth muscle fibers to reveal the molecular arrangement of the endomembrane system (referred to variously in the text as ER or sarcoplasmic reticulum [SR]; S-ER or ER/SR) known to participate in the control of Ca2+ homeostasis. The lumenal ER chaperon, immunoglobulin binding protein (BiP), as well as protein disulfide isomerase, and calreticulin, a Ca2+ binding protein expressed by most eukaryotic cells, appeared to be evenly distributed throughout the entire system (i.e., within [a] the nuclear envelope and the few rough-surfaced cisternae clustered near the nucleus; [b] single elements scattered around in the contractile cytoplasm; and [c] numerous, heterogeneous, mainly smooth-surfaced elements concentrated in the peripheral cytoplasm, part of which is in close apposition to the plasmalemma). All other structures, including nuclei, mitochondria, Golgi complex, and surface caveolae were unlabeled. An even distribution throughout the endomembrane system appeared also for the proteins recognized by anti-ER membrane antibodies. In contrast, calsequestrin (the protein that in striated muscles is believed to be the main actor of the rapidly exchanging Ca2+ storage within the lumen of the sarcoplasmic reticulum) was found preferentially clustered at discrete lumenal sites, most often within peripheral smooth-surfaced elements of moderate electron density. Within these elements dual labeling revealed intermixing of calsequestrin with the other lumenal ER proteins. Moreover, the calsequestrin-rich elements were enriched also in the receptor for inositol 1,4,5-trisphosphate, the second messenger that induces Ca2+ release from intracellular stores. These results document the previously hypothesized molecular heterogeneity of the smooth muscle endomembrane system, particularly in relation to the rapid storage and release of Ca2+.

Animals↗
PubMed1993

Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7.

ABP-280 is a ubiquitous actin binding protein present in the cytoskeleton of many different cell types. ABP-280 was mapped to distal Xq28, 50-60 kb downstream of the Green Colour Pigment (GCP) genes. To establish if ABP-280 may be a candidate for one of the muscle disease localized by linkage analysis to distal Xq28 we looked for alternative forms of ABP-280 mRNA. Several different ABP-280 mRNAs were indeed identified: two are X-linked and are produced by alternative splicing of a small exon of 24 nucleotides. At least one additional gene encoding a RNA more than 70% identical to ABP-280 in the 1700 bp sequenced has also been found. It was mapped to chromosome 7. While both forms of the X-linked ABP-280 are ubiquitous, the gene on chromosome 7 is highly expressed only in skeletal muscle and heart. The two genes were therefore excellent candidates for the X-linked and for the autosomal dominant form of the Emery-Dreifuss Muscular Dystrophy (EDMD) both of which have been described. So far, however we were unable to demonstrate mutations in the coding region or affecting the alternative splicing of the X-linked form of ABP-280, in several patients studied, and we think that it is quite unlikely that this is the gene responsible for EDMD.

Amino Acid Sequence↗
PubMed1993

Fluoride absorption from disodium and calcium monofluorophosphates from the gastrointestinal tract of rats.

The contribution of the stomach and the small intestine to the in vivo absorption of fluoride from disodium and calcium monofluorophosphates was studied in comparison with that observed when sodium fluoride is given to rats. In vitro experiments with ligated duodena were also carried out. Fasted rats were given orally 50 micrograms of fluoride in pH 6.3 buffered disodium monofluorophosphate solutions with and without 50 mM added calcium. The same amount of fluoride was administered in the form of an aqueous sodium fluoride solution. In both cases the fluoride solutions contained trace amounts of (14C)-labeled polyethyleneglycol as a marker for the study of water movement through the gastrointestinal tract. Results obtained show the small intestine as the main site of the absorption of the studied compounds. Under the fasting conditions employed in our study gastric absorption was in turn, almost negligible. While fluoride transport from sodium fluoride seems to occur through a difussion mechanism, disodium and calcium monofluorophosphates appear to be absorbed through a more complex mechanism involving an enzymatic hydrolysis and an independent and possibly parallel direct tissue absorption of a soluble, neutral calcium monofluorophosphate complex.

Administration, Oral↗
PubMed1993

Evolution of fluoride concentrations in cattle and grass following a volcanic eruption.

This study determined fluoride concentrations of forage, water and bovine serum 2-y after the cessation of a volcanic eruption. Three farms within the polluted area were selected, and water and green grass were collected from April 1990 to February 1992. Samples of hay were collected twice from each farm. Five cows exposed during the 13-mo eruption and 6 young bulls not exposed during the volcanic eruption had blood collected monthly or bimonthly. Serum fluoride concentrations and alkaline phosphatase activities were determined and dental conditions observed in all animals. Grass fluoride remained low except during spring of the second year; high fluoride was in the hay. Serum fluoride of the cows remained near acceptable reference values, although some cows had higher values; serum fluoride of the bulls markedly increased during the spring of the second year. Severe incisor attrition occurred in the cows, while the young bulls had less severely eroded teeth.

Air Pollutants↗
PubMed1993

[The remodelling of the heart in heart failure: from thoracic radiography to magnetic resonance].

Congestive heart failure represents the most common medical hospital discharge diagnosis, and can occur in patients with preserved indexes of left ventricular systolic function, even in absence of patent coronary or valvular heart disease. The present review examines the role of imaging techniques in the diagnosis and follow-up of these patients. Imaging of the heart has undergone dramatic advances with the development and refinement of new imaging modalities such as echocardiography, computed tomography, magnetic resonance and radionuclide emission tomography. The role of "low-tech" modalities such as chest roentgenogram is discussed. The possibilities offered by ultrasounds or magnetic resonance in tissue characterization are then compared with the actual capability of cardiac imaging in detecting myocardial tissue alterations (oedema, ischemia, myocarditis, etc.) and/or degeneration (fatty degeneration, fibrosis, amyloidosis, etc.). Finally, the potential use in modern clinical medicine of magnetic resonance spectroscopy and positron emission tomography to study myocardial metabolism and cellular function are discussed.

Angiocardiography↗
PubMed1993

[Dynamic vascular imaging with magnetic resonance imaging of the thoracic and abdominal region. Technique optimization].

Several Magnetic Resonance (MR) imaging techniques for the study of the main thoracic and abdominal vessels are analyzed. Such techniques based on the static representation of vessels, as MR angiography (MRA), are considered, together with dynamic techniques--i.e., cine MR--and those based on ultra-fast sequences with bolus contrast medium administration; the latter are considered also according to their use in the study of the early parenchymogram. Namely, the investigated techniques are: 3D/2D inflow imaging with and without presaturation, 3D inflow imaging with paramagnetic contrast medium administration, 2D/3D phase/dephase subtraction imaging, cine MR with heart gating, the sequential dynamic single-slice technique with bolus contrast medium, and the apnea multi-slice imaging. The main parameters are indicated for each technique and type of sequence. From our experience, rather precise indications emerge as to the use of the various techniques according to the investigated region and to the suspected disease. The best techniques for demonstrating sacciform aneurysms proved to be the 3D inflow ones, as well as the cine MR and the turbo-flash sequences with contrast medium; as for dissecting aneurysms, cine MR proved best. In portal flow conditions and in major veins thromboses, 2D inflow and phase/dephase subtraction sequences are suggested. In the study of renal stenoses, limitations and advantages of 2D versus 3D sequences are compared. Moreover, indications, limitations and specificity are analyzed of the early parenchymogram based on ultra-fast sequences with paramagnetic contrast medium. In the authors' experience, the different MR vascular imaging techniques must be considered only an integration to more specific investigations, but it is likely that, as it happened with MRA of the head and neck, the increase in resolution and the reduction in artifacts will--soon--turn this kind of imaging into the examination of choice in vascular studies.

Abdomen↗
PubMed1993

[Dynamic sequential study of hepatic nodular lesions with ultra-fast sequences and fast bolus of gadolinium-DTPA].

Eighty-five patients with single/multiple nodular hepatic lesions (10 focal nodular hyperplasias, 37 liver hemangiomas, 24 metastases and 16 hepatocellular carcinomas; 2 patients had associated lesions) were examined with dynamic single-slice sequences and fast i.v. bolus injection of Gd-DTPA. The dynamic single-slice technique was used to evaluate the peculiar features of the dynamic enhancement. The snapshot sequence proved best to provide the high temporal resolution of the dynamic parenchymal enhancement (scanning time < 1 second, one frame every third second). The following variables were investigated: nodular lesion intensity in the first basal snapshot image, enhancement appearance and its contemporaneity with arterial, venous or portal flows, enhancement gradient relative to surrounding liver parenchyma, morphologic features of the enhancement and its centrifugal/centripetal patterns. The enhancement curve of focal nodular hyperplasia increased very quickly during the first 20-25 seconds. This enhancement was quite similar to the arterial one and always occurred before portal and systemic venous times. Hemangiomas exhibited a typically slow growth-curve in the first 120 seconds, with a positive final gradient value relative to liver parenchyma. The appearance of peripheral contrast enhancement was a typical sign after 30 seconds. Metastases exhibited similar dynamic enhancement to hemangiomas, but peripheral enhancement was never observed and, after 120 seconds, gradient was null or negative relative to the adjacent liver parenchyma. Moreover, enhancement always followed portal times. Hepatocellular carcinomas showed an early growth curve, preceding portal times, but less marked than in hyperplasia. The study of our series provided the preliminary semiology of early dynamic enhancement patterns, which is quite specific to recognize and differentiate nodular hepatic lesions.

Contrast Media↗
PubMed1992

A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

Amino Acid Sequence↗
PubMed1992

Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice.

Transgenic mice carrying various oncogenes driven by mammary gland specific enhancers develop mammary tumors usually arising in a stochastic way. The only exception is a mouse lineage (TG.NF) carrying an activated rat Neu oncogene driven by the murine mammary tumor virus long terminal repeat (MMTV-LTR) that gave rise to rapid and multifocal mammary tumors interpreted as a result of a single-step neoplastic transformation. The effect of the oncogene appeared to be specific for breast tissue, since salivary and Harderian glands as well as epididymis expressed high levels of Neu but only developed hyperplasia (Muller et al., Cell, (1988) 54, p. 105). Here we describe a transgenic mouse lineage for the MMTV-Neu, analysed up to third generation. Multifocal tumors involving mammary glands arose very rapidly in all females independently from pregnancy and in some males. Moreover, multifocal neoplasias occurred also in salivary and Harderian glands and in the epididymis at a very high rate. These data demonstrate that the Neu oncogene can induce tumors in all the tissues where it is expressed at high levels.

Animals↗
PubMed1992

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.

Animals↗