Biomedical subjects
M Shani
Publications and source records attributed to M Shani.
Application of germline transformation to the study of myogenesis.
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[Changing economic environment of hospitals: management challenges of the 1990s].
The modern hospital is an organization which is influenced by the external environment in which it functions. A major relevant area is the economic environment. In recent years the western world has been facing the challenge of rising costs of health care and an increase in their proportion to the gross national product of most countries. Consequently, hospitals as major providers of health care are under pressure from governments and health insurance companies to cut costs and to "produce" more efficiently. Since hospitals worldwide are finding it hard and painful to function in the new environment in which attitudes to hospitals are changing, a potential managerial-economic crisis may be the next phase. How can the hospital adapt to these changes? First, by adopting managerial attitudes and the tools of the business sector. These include: the strategic planning process, hospital operative autonomy, creating medical-economic responsibility centers as departments, cost-accounting for medical procedures, and case-mix budgeting. Management information systems are necessary during the transition. The hospital information system should include functions at the operative level, such as outpatient visits, and admissions and discharges of patients; and also clinical, diagnostic and laboratory procedures related to the patient case-mix. The second level is a management information system which includes salaries of personnel, case-mix budgeting with variance analysis, prices of procedures and epidemiological data. The authors believe that only the managerial approach combining medical and economic disciplines can meet the challenges of the changing modern economic environment.
Modulation of chick intestinal and renal calbindin gene expression by dietary vitamin D3, 1,25-dihydroxyvitamin D3, calcium and phosphorus.
Synthetic oligonucleotide probes complementary to chick calbindin-28 kDa-mRNA were used to study the latter's regulation and relationship to calbindin in the chick. The effects of vitamin D3 sources and dietary alteration on the genomic expression were characterized by Northern blot and solution hybridization. Intestinal calbindin and its mRNA were almost absent in vitamin D-deficient chicks and were not affected by dietary alteration. Renal calbindin and its mRNA were lower in the vitamin D-deficient than in vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-fed chicks. In the same animal, renal calbindin mRNA and calbindin were higher than intestinal. In vitamin D3-fed chicks, dietary calcium (Ca) or phosphorus (P) restriction induced, and high dietary Ca inhibited, intestinal calbindin and its mRNA synthesis. In the same chicks, dietary P restriction induced renal calbindin mRNA and calbindin synthesis. In 1,25-(OH)2D3-fed chicks, dietary P restriction induced and high dietary Ca inhibited the synthesis of intestinal and renal calbindin. The results suggest that: (a) most of the changes in renal and intestinal calbindin could be attributed to the changes in the mRNA; (b) the adaptation to dietary Ca and P alterations requires vitamin D metabolites; (c) high dietary Ca affects intestinal and renal calbindin-mRNA and calbindin via mechanisms independent of kidney 1-hydroxylase; and (d) plasma Ca and renal calbindin or its mRNA tend to change together in vitamin D-deficient or vitamin D3-fed, but not in 1,25(OH)2D3-fed chicks.
Demethylation of genes in animal cells.
Tissue-specific animal cell genes are usually fully methylated in the germ line and become demethylated in those cell types in which they are expressed. To investigate this process, we inserted a methylated IgG kappa gene into fibroblasts and lymphocytes at various stages of development. The results show that this gene undergoes demethylation only in the mature lymphocytes and therefore suggest that the ability to demethylate a gene is developmentally regulated. These studies were supported by similar experiments using the rat Insulin I gene, and in this case it appears that the cis-acting elements that control demethylation may be different from those responsible for gene activation. The ability to demethylate the housekeeping gene APRT is also under developmental control, because this occurs only in embryonic cells, both in tissue culture and in transgenic mice.
Amplification of mineralocorticoid activity of aldosterone by 18-hydroxy-corticosterone and 18-hydroxy-19-nor-corticosterone in adrenalectomized rats.
A combination of aldosterone (1 micrograms) with either 18-OH-corticosterone (1 micrograms) or 18-OH-19-norcorticosterone (1 micrograms) injected to adrenalectomized rats indicated an amplification of mineralocorticoid activity as expressed by Na/K ratio in urine. Without aldosterone their mineralocorticoid potency was negligible.
The amount of the endogenous and exogenous skeletal muscle actin mRNA in the heart of transgenic mice is affected by the genotype of the cardiac actin gene.
Both skeletal muscle and cardiac actins are co-expressed in the newborn heart. However, the amount of the skeletal muscle actin and its mRNA rapidly decreases during early development and the cardiac actin predominates in the adult heart. In BALB/c and DBA mice there is a mutation in the cardiac actin gene which is associated with decreased levels of cardiac actin mRNA and high levels of the skeletal muscle actin transcript in the adult heart. To examine the possibility that the amount of cardiac actin gene product modulates the expression of the skeletal muscle actin gene in the heart, transgenic mice carrying a tagged skeletal muscle actin gene were produced, and the expression of the endogenous and endogenous and exogenous actin gene was analyzed in offspring carrying different combinations of the cardiac actin alleles. It was found that both the endogenous and exogenous skeletal muscle actin genes were expressed at low levels in the heart of adult mice homozygous for the wild-type cardiac actin gene allele, at abnormally high levels in mice homozygous for the mutated cardiac actin allele, and at intermediate levels in heterozygous mice. This shows that the level of expression of the cardiac actin gene has a trans effect on the expression of the skeletal muscle actin gene.
Do we really spend enough on health services in Israel?
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Analysis of muscle-specific gene expression by germ line transformation approaches.
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Regulated expression of muscle-specific genes introduced into mouse embryonal stem cells: inverse correlation with DNA methylation.
Pluripotent embryonal stem cell lines (ES) were isolated from cultured normal mouse blastocysts. These cells retained their capacity to differentiate into a great variety of cell types in cell cultures or in tumors formed after subcutaneous injection of the cells into nude mice. A chimeric actin/globin gene containing about two-thirds of the rat skeletal muscle actin gene and 730 bp of its 5' flanking region fused to the 3' end of the human embryonic epsilon-globin gene, was inserted into a plasmid containing a neomycin resistance gene (neor) whose transcription is regulated by the SV40 early control elements. The prokaryotic vector DNA sequences of this plasmid (pAG-Neo) were deleted and the two linked genes were introduced into the ES cells by electroporation. G418-resistant clones were isolated, amplified and injected subcutaneously into nude mice. From the teratocarcinoma-like tumors which developed we isolated myogenic as well as nonmyogenic cell lines. In cell lines derived from three independent transfected ES clones, expression of the actin/globin gene was developmentally regulated in myogenic cells. In contrast, in a number of experiments in which the actin/globin gene or other muscle-specific genes were introduced into the ES cells without the removal of the pBR sequences, no expression could be detected at any stage. Moreover, in the differentiated lines derived from these clones, G418 resistance was lost, and no neor transcripts could be detected. Southern-blot analysis of MSPI- or HpaII-digested DNA revealed extensive methylation in the clones that did not express the foreign DNA, whereas no significant methylation of the inserted DNA was observed in clones which expressed the transfected genes. Examination of the DNA extracted from transgenic mice carrying the same actin/globin gene revealed an inverse correlation between methylation of the exogenous gene and its potential to be expressed in the transgenic strain. However, no tissue-specific differences in methylation, related to the tissue specificity of expression of the exogenous gene, could be detected in these experiments. These results suggest that the process of methylation reported here is causally related to constitutive inactivation of the exogenous genes.
The hypertensinogenic activity of 18-hydroxy-19-norcorticosterone in the adrenalectomized rat.
Hypertensinogenic properties of recently synthesized 18-OH-19-nor-B and the related 18-OH-B were examined in adrenalectomized, spontaneously hypertensive rats (SHR). Each steroid was given via Alzet miniosmotic pump (2002), with and without aldosterone. Neither 18-OH-19-nor-B (5 micrograms) nor 18-OH-B (5 micrograms) increased blood pressure in SHR when given alone, but when administered together with aldosterone (5 micrograms), which was ineffective by itself, resulted in a significant rise in blood pressure. There is a high probability that certain mineralocorticoids, which are inactive by themselves, are of importance in the etiology of hypertension under physiological conditions.
Expression of the rat myosin light-chain 2 gene in transgenic mice: stage specificity, developmental regulation, and interrelation with the endogenous gene.
The expression of the rat skeletal myosin light-chain 2 gene in two transgenic strains was tissue specific and stage specific. However, the temporal regulation during development of the transgene was different from that of the endogenous gene. Surprisingly, in one strain, the expression of the transgene was associated with a significant down-regulation of the endogenous gene. The possible mechanisms to account for the suppression of the endogenous gene and the potential implications of this suppression are discussed.
Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay.
This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na+ + K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K0.5 100-200 microM; plus ATP, K0.5 0.8-1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E(1)2Rb+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30-100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, kobs = 0.08 s-1, in the presence of either Rb+ (20 mM) or Na, (100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique. (Na+ + K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na+ + K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E1-E2 conformational transition of the unphosphorylated protein probably remains.
Expression in transgenic mice of two genes of different tissue specificity integrated into a single chromosomal site.
Transgenic mice were used to study the expression of pairs of genes with distinctly different tissue specificities, covalently linked and integrated into the same chromosomal site. A transgenic strain carrying, in close proximity and in the same orientation, the rat fast skeletal muscle myosin light-chain 2 (MLC2) gene and the mouse rearranged immunoglobulin kappa light-chain gene expressed the immunoglobulin gene specifically in the lymphoid tissues, whereas rat MLC2 transcripts were found in skeletal muscle but not in the spleen or the other tissues that were tested. In another transgenic strain, carrying the rat MLC2 gene and a modified rat skeletal muscle actin gene (actin-globin chimeric gene), transcripts of the rat MLC2 gene were detected in skeletal muscle only, whereas the actin-globin transcripts were detected in skeletal muscle as well as in the heart. Moreover, the expression of the chimeric gene was also developmentally regulated. Expression was higher in cardiac muscle than in the skeletal muscle of neonatal mice, whereas expression was higher in skeletal muscle in adult mice. This pattern is consistent with the regulation of the expression of the endogenous skeletal muscle actin gene. Thus, in those transgenic strains that expressed both genes, each gene retained its tissue specificity, in spite of their close proximity. These results indicate a high degree of autonomy of the control elements included in the cloned genomic DNA fragment and demonstrate that a single chromosomal site can be permissive for the proper expression of two genes with different tissue specificities.
Renovascular hypertension in spontaneous hypertensive rats: an experimental model of renal artery stenosis superimposed on essential hypertension.
Renovascular hypertension superimposed on essential hypertension, a condition encountered in the elderly, was studied. An experimental animal model consisting of a two-kidney one-clip Goldblatt preparation in the spontaneous hypertensive (SHR) rat, that would simulate this condition, was designed. A 0.25 mm silver clip was placed on the left renal artery of SHR male rats. The same procedure performed on WKY rats served as control. All experiments were performed on low, normal, and rich sodium diet. Systolic blood pressure (BP) was measured by tail-cuff method. Plasma renin concentration (PRC) was determined before and after clipping of the renal artery. Results were as follows: Mean systolic BP increased significantly in clipped rats fed with normal and rich sodium diets. SHR showed an increase from 144 +/- 3 (mean + s.e.m.) to 168 +/- 3 mmHg, and WKY rats showed an increase from 120 +/- 2 to 139 +/- 5 mmHg. There was a two- to threefold rise in PRC. A low-salt diet given prior to clipping prevented the appearance of renovascular hypertension despite a significant rise in PRC. We concluded that renal artery narrowing plays a significant role in the rise of BP in the basically essential type of hypertension.
Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.
A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.
The nucleotide sequence of a rat myosin light chain 2 gene.
A rat myosin light chain 2 gene was characterized by nucleotide sequence and S1 mapping analyses. It contains seven exons separated by six introns. The corresponding mRNA is predicted to be 654 nucleotides long (excluding polyA sequences), with 5'-nontranslated, coding, and 3'-nontranslated lengths of 56, 510, and 88 nucleotides, respectively. The predicted amino acid sequence is identical to that from rabbit except that the rat sequence lacks one of two Gly residues located at positions 12 and 13 in the rabbit sequence. From the nucleotide sequence, nascent rat myosin light chain 2 is predicted to have Met Ala preceding Pro at the N-terminal end.
The methylation state of 2 muscle-specific genes: restriction enzyme analysis did not detect a correlation with expression.
To examine the possible role of DNA methylation in the modulation of expression of genes involved in the differentiation of muscle cells, we compared the methylation state of a number of CpG sites in the rat skeletal muscle actin and myosin light chain 2 genes, in muscle and nonmuscle cells, and in proliferating myoblasts and differentiated myotubes of the myogenic cell line L8. No correlation was detected between the state of methylation of these sites and the expression of the two genes. Essentially the same pattern of DNA methylation was observed, in the sites examined, in DNA from muscle, kidney and stomach. In DNA extracted from cultures of proliferating mononucleated myoblasts, as well as from differentiated multinucleated fibers of the myogenic cell line L8, the two genes were more methylated than in other tissues.