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Biomedical subjects

M Shani

Publications and source records attributed to M Shani.

At least 19 recordsLinked to original sources
PubMed2001

Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome.

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A-->T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G-->A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein.

Biological Transport↗
PubMed2001

A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells.

A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis.

Animals↗
PubMed2001

The two PDGF receptors maintain conserved signaling in vivo despite divergent embryological functions.

Gene targeting studies have indicated that the two receptors for PDGF, alpha and beta, direct unique functions during development. Distinct ligand affinities, patterns of gene expression, and/or mechanisms of signal relay may account for functional specificity of the two PDGF receptor isoforms. To distinguish between these factors, we have created two complementary lines of knockin mice in which the intracellular signaling domains of one PDGFR have been removed and replaced by those of the other PDGFR. While both lines demonstrated substantial rescue of normal development, substitution of the PDGFbetaR signaling domains with those of the PDGFalphaR resulted in varying degrees of vascular disease. This observation provides a framework for discussing the evolution of receptor tyrosine kinase functional specificity.

Animals↗
PubMed2000

The impact of information on medical thinking and health care policy.

The information revolution has, till now, concentrated on the collection, storage, transfer, and analysis of data, with emphasis on the technology and not on the information itself. Information in health care is conductive to two major changes. One aspect is the conversion of clinical medicine from an art, based on clinical intuition to a more scientific profession based on clinical guidelines and evidence based medicine. The second aspect relates to health care policy based on better information.

Clinical Medicine↗
PubMed1999

Manipulations of ACHE gene expression suggest non-catalytic involvement of acetylcholinesterase in the functioning of mammalian photoreceptors but not in retinal degeneration.

To explore role(s) of acetylcholinesterase (AChE) in functioning and diseased photoreceptors, we studied normal (rd/+) and degenerating (rd/rd) murine retinas. All retinal neurons, expressed AChEmRNA throughout fetal development. AChE and c-Fos mRNAs peaked at post-natal days 10-12, when apoptosis of rd/rd photoreceptors begins. Moreover, c-Fos and AChEmRNA were co-overexpressed in rd/rd mice producing transgenic human (h), and host (m) AChE, but not in rd/+ mice. However, mAChE overexpression also occurred in transgenics expressing human serum albumin. Drastic variations in AChE catalytic activity were ineffective during development. Neither transgenic excess nor diisopropylfluorophosphonate (DFP) inhibition (80%) affected the rd phenotype; nor did DFP exposure induce photoreceptor degeneration or affect other key cholinergic proteins in rd/+ mice, unlike reports of adult mice and despite massive induction under DFP of c-Fos70 years). Therefore, the extreme retinal sensitivity to AChE modulation may reflect non-catalytic function(s) of AChE in adult photoreceptors. These findings exclude AChE as causing the rd phenotype, suggest that its primary function(s) in mammalian retinal development are non-catalytic ones and indicate special role(s) for the AChE protein in adult photoreceptors.

Acetylcholinesterase↗
PubMed1999

In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with beta-lactoglobulin and whey acidic protein promoters.

The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern of HSA expression in both WAP/HSA and BLG/HSA transgenic lines was copy number independent, and the major site of ectopic expression was the skeletal muscle. Although an equal proportion of expressors was determined in both sets of mice (approximately 25% secreting >0.1 mg/ml), the highest level of HSA secreted into the milk in the WAP/HSA transgenic lines was one order of magnitude lower than in the BLG/HSA lines. Despite this difference, the HSA expression patterns in the mammary gland were similar and consisted of two levels of variegated expression. Studies using mammary explant cultures revealed a comparable responsiveness to the lactogenic hormones insulin, hydrocortisone, and prolactin, although the WAP/HSA gene constructs were more sensitive to the hydrocortisone effect than were the BLG/HSA vectors. When HSA vectors were stably transfected into the mouse mammary cell line CID-9, they displayed a hierarchy of expression, dependent upon the specific complement of HSA introns included. Nevertheless, the expression of HSA in four out of five WAP/HSA constructs was similar to their BLG/HSA counterparts. This construct-dependent, and promoter-independent, hierarchy was also found following transfection into the newly established Golda-1 ovine mammary epithelial cell line.

Animals↗
PubMed1998

Establishment and initial characterization of the ovine mammary epithelial cell line NISH.

Analysis of the molecular mechanisms involved in the differentiation and formation of the characteristic three-dimensional structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete beta-lactoglobulin (BLG) in an ECM (extracellular matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously needed to maintain BLG expression. Stable transfection of NISH cells with BLG/Human Serum Albumin (HSA) hybrid gene constructs revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in cells transfected with hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and organogenesis of mammary epithelial cells under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for the transgenic mouse model in evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals.

Animals↗
PubMed1998

Insulin and prolactin synergize to induce translation of human serum albumin in the mammary gland of transgenic mice.

A dramatic uncoupling of the expression of chimaeric beta-lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium. HSA RNA continued to accumulate, whereas the synthesis of the corresponding protein was dependent on the presence of insulin and prolactin with a minor contribution of hydrocortisone. The untranslated HSA RNA was indistinguishable from its translatable counterpart in its mobility on agarose gels, was transported normally from the nucleus to the cytoplasm and was translated efficiently in rabbit reticulocyte lysate. In the presence of cycloheximide, HSA RNA rapidly disappeared suggesting a dependency on ongoing protein synthesis. Its estimated half-life of 5-6 h in hormone-free medium increased significantly in the presence of insulin, hydrocortisone and prolactin and was comparable to that of beta-casein RNA. The uncoupling of the expression of the BLG/HSA transgenes at the RNA and protein levels was also confirmed by in situ hybridization and immunohystochemistry on sections from virgin mammary explants. HSA synthesis was initiated within 13 h of the addition of insulin and prolactin in explants that had accumulated untranslated HSA RNA and was fourfold higher than that observed with insulin alone. Addition of hydrocortisone contributed to an additional 20% in HSA synthesis. We believe this is the first demonstration of translational control of exogenous milk protein gene expression in the mammary gland of transgenic animals.

Animals↗
PubMed1997

[The changing world].

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Computer Communication Networks↗
PubMed1997

Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin Ibeta gene expression followed by late-onset neuromotor deterioration.

To explore the possibility that overproduction of neuronal acetylcholinesterase (AChE) confers changes in both cholinergic and morphogenic intercellular interactions, we studied developmental responses to neuronal AChE overexpression in motoneurons and neuromuscular junctions of AChE-transgenic mice. Perikarya of spinal cord motoneurons were consistently enlarged from embryonic through adult stages in AChE-transgenic mice. Atypical motoneuron development was accompanied by premature enhancement in the embryonic spinal cord expression of choline acetyltransferase mRNA, encoding the acetylcholine-synthesizing enzyme choline acetyltransferase. In contrast, the mRNA encoding for neurexin-Ibeta, the heterophilic ligand of the AChE-homologous neuronal cell surface protein neuroligin, was drastically lower in embryonic transgenic spinal cord than in controls. Postnatal cessation of these dual transcriptional responses was followed by late-onset deterioration in neuromotor performance that was associated with gross aberrations in neuromuscular ultrastructure and with pronounced amyotrophy. These findings demonstrate embryonic feedback mechanisms to neuronal AChE overexpression that are attributable to both cholinergic and cell-cell interaction pathways, suggesting that embryonic neurexin Ibeta expression is concerted in vivo with AChE levels and indicating that postnatal changes in neuronal AChE-associated proteins may be involved in late-onset neuromotor pathologies.

Acetylcholinesterase↗
PubMed1996

Beta-lactoglobulin/human serum albumin fusion genes do not respond accurately to signals from the extracellular matrix in mammary epithelial cells from transgenic mice.

Mammary epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously, we demonstrated that non-HSA-secreting transgenic strains retain the potential to express the HSA transgene in vitro and that mammary epithelial cell cultures from non-HSA-secreting strains express higher levels of HSA when grown on tissue culture plastic than they do when grown on collagen. In this study we studied the expression of BLG/HSA fusion genes in epithelial cell cultures of additional transgenic strains and additional substrata. Our results show that: (1) The BLG/HSA fusion gene in only one of seven HSA-secreting or nonsecreting transgenic strains tested accurately responded to signals from the EHS matrix; (2) HSA DNA sequences dominantly affected the activity of BLG as well as the whey acidic protein promoters; and (3) HGF/SF induced both milk proteins and HSA gene expression. These results suggest that the response to the extra cellular matrix (ECM) plays a key role in the expression of BLG/HSA fusion genes and that the function of the regulatory elements within the promoter regions of milk protein genes involved in response to the ECM, in developmental and in tissue specificity, very much depend on the downstream gene sequences.

Animals↗
PubMed1996

Dual regulation of beta-lactoglobulin/human serum albumin gene expression by the extracellular matrix in mammary cells from transgenic mice.

Mammary explants and epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously we demonstrated that mammary explants from virgin female transgenic mice synthesize and secrete high levels of HSA during the first day in culture. Here we present a detailed analysis of endogenous and transgene expression during the first 20 h of mammary explant cultures. We show that HSA genes as well as endogenous milk protein genes are rapidly induced upon explantation. Unexpectedly, HSA was synthesized also in mammary explants from strains that do not secrete HSA into the milk, indicating the existence of a cryptic potential to express the transgene. Histological examination revealed that some luminal epithelial cells detached from the underlying extracellular matrix (ECM) soon after explantation. Epithelial cell cultures from nonsecreting strains grown on plastic rapidly induced transgene expression and secreted higher levels of HSA into the medium compared to cells grown on collagen. These results suggest that tissue organization and most likely the interaction of epithelial cells with the ECM are intimately involved in the control of HSA transgene expression.

Animals↗
PubMed1996

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.

Animals↗
PubMed1996

Co-integration of beta-lactoglobulin/human serum albumin hybrid genes with the entire beta-lactoglobulin gene or the matrix attachment region element: repression of human serum albumin and beta-lactoglobulin expression in the mammary gland and dual regulation of the transgenes.

The effect of co-integration of the entire beta-lactoglobulin (BLG) gene or matrix attachment region (MAR) sequences on the expression of various BLG/ human serum albumin (HSA) gene constructs was tested in transgenic mice. These former sequences were chosen because of their reported ability to insulate transgenes from the neighboring host genomic DNA sequences and/or to provide a more permissive transcriptional environment. When introduced alone, a cDNA-based BLG/HSA construct was expressed in 60% of transgenic strains and HSA was secreted at levels up to 0.3 mg/ml into the milk. Upon co-integration with either the entire BLG gene or MAR element, HSA RNA and protein expression were completely abrogated. While the co-integrated BLG gene suppressed the proportion of expresser strains carrying cDNA as well as genomic BLG/HSA constructs, the MAR element only exerted its negative effect on the cDNA-based BLG/HSA construct. In transgenics expressing both HSA and BLG, the tissue specificity and developmental patterns of BLG expression were altered and resembled the less stringent pattern of the BLG/HSA expression. These results demonstrate that rescue of transgene expression through co-integration with BLG or MAR sequences do not apply universally.

Animals↗