Dual regulation of expression of exogenous delta-crystallin gene in mammalian cells: a search for molecular background of instability in differentiation.
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Animals↗
Biomedical subjects
H Kondoh
Publications and source records attributed to H Kondoh.
In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.
Expression of the chicken delta-crystallin gene 1 injected into the nuclei of mouse cells is lens specific. Coinjection of GC box-containing DNA fragments from delta-crystallin, simian virus 40 early, and herpes simplex virus type 1 tk promoters effectively suppressed delta-crystallin expression in the lens, but coinjection with DNA fragments not containing the GC box did not. This suppression was likely due to the competition of an Sp1-like transcription factor(s) and indicates involvement of the apparently ubiquitous factor(s) in the tissue-specific expression of the delta-crystallin gene.
Animals↗
Effect of concanavalin A on the aspirin concentration and distribution in the brain and plasma of rats.
The effects of concanavalin A (Con A) on plasma and brain concentration of aspirin (Asp) were investigated in rats. When Asp was administered in rats treated with Con A, the plasma and brain concentration of Asp and its metabolites (salicylic acid (SA), salicyluric acid (SU) and gentisic acid (GA)) were increased. Distribution of Asp and its metabolites into the hippocampus, striatum and hypothalamus were increased by treatment with Con A. Asp esterase activities in the small intestinal mucosa, liver and brain were increased by pretreatment with Con A, but these enzyme activities were decreased by a high dose of Con A. The inhibitory effect of Asp, SA, SU and GA on the writhing induced by acetic acid in Con A-pretreated mice was stronger than that in the control. A lowering of rectal temperature, after the administration of Asp, was stronger in Con A-treated rats than in control rats. These results suggested that Con A facilitated the absorption of Asp and enhanced the transfer of SA into the brain, and increased Asp esterase activities in various tissues.
Analgesics↗
Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region.
A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation.
Amino Acid Sequence↗
DNA sequences responsible for tissue-specific expression of a chicken alpha-crystallin gene in mouse lens cells.
We have studied the DNA sequences required for high-level expression of a cloned chicken alpha-crystallin gene by introducing a hybrid alpha/delta-crystallin gene into nuclei of mouse lens epithelial cells in primary culture. The level of transient expression of the hybrid gene consisting of the 5' upstream promoter region of the alpha-crystallin gene fused to the structural portion of the delta-crystallin gene was determined by Western blot analysis using anti-delta-crystallin serum. The hybrid gene appears to be expressed in a tissue-specific manner, since it is active in mouse lens cells but not in fibroblasts or in L cells. The DNA sequences located 242-189 bp upstream from the transcription initiation site are required for high-level expression in lens cells. They are active when their orientation is reversed at the original site or when placed approximately 1.7 kbp downstream from the cap site in the second intron of the hybrid gene in either orientation. When these DNA sequences were replaced by the enhancer sequences of Moloney murine leukemia virus, the hybrid gene was expressed in both lens cells and fibroblasts.
Animals↗
Arachidonic acid metabolism in cultured mouse keratinocytes.
We attempted to characterize the general features of arachidonate metabolism in cultured mouse keratinocytes. The cells labeled with [3H]arachidonate were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), ionophore A23187, and fetal bovine serum (FBS). Common to the three substances, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine almost equally served as sources of arachidonate liberated by the action of phospholipase A2. The stimulation of phospholipase A2 action was observed in the order of A23187 greater than FBS greater than TPA. When stimulated by TPA or A23187, the radioactivity released into the extracellular medium was mostly found in prostaglandin (PG) E2. Formation of other PGs and hydroxyeicosatetraenoate (HETE) was extremely limited. In the case of stimulation by FBS, however, the released radioactivity was mainly associated with non-converted arachidonate. FBS also inhibited the TPA- and A23187-induced conversion of arachidonate to PGE2. Phospholipid degradation induced by the three stimulators was similarly dependent on extracellular Ca2+. The stimulation by FBS and A23187 was suppressed by calmodulin antagonists, though the effect of A23187 was much more sensitive to the antagonists when compared to that of FBS. We observed more than additive effects of the three stimulators when tested together.
Animals↗
[Involvement of the 5' end region in the tissue-specific regulation of a chicken delta-crystallin gene].
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Animals↗
Cyclic sinus node dysfunction in a patient with hyperthyroidism.
In a 54-year-old woman with hyperthyroidism, episodic sinus node dysfunction (SND) recurred in a cyclic fashion. The SND occurred suddenly during normal sinus rhythm and progressed to marked sinus bradycardia; occasionally, sinus arrest developed with or without ventricular escape beats. A 1-mg dose of intravenous atropine sulfate suppressed the occurrence transiently. After a 200-mg dose of propylthiouracil and a 20-mg dose of prednisolone every six hours, the SND was completely abolished within 21 hours. It is probable that, together with a pathologic process induced by hyperthyroidism, the autonomic nervous system participated in our patient's episodic SND.
Arrhythmias, Cardiac↗
Differentiation-dependent expression of the chicken delta-crystallin gene introduced into mouse teratocarcinoma stem cells.
PCC3 mouse teratocarcinoma (TCC) stem cells were cotransfected with either the plasmid p delta C-1A or p delta C-1B carrying the chicken delta-crystallin gene, and with the plasmid pSV2gpt containing the selectable bacterial xanthine-guanine phosphoribosyltransferase (XGPRT) gene, using the calcium phosphate technique. Nine transformed PCC3 stem cell lines, each of which was clonally derived from respective colonies surviving after the selection process, were isolated. Southern blot analysis revealed that all of them stably maintained delta-crystallin sequences associated with high mol. wt. cellular DNA after propagation in non-selective medium in vitro, and after the production of solid tumors in the syngenic host mice. Six cell lines contain the intact delta-crystallin gene sequence and eight contain the gpt sequence. The number of delta-crystallin DNA copies was highly variable among transformed lines, 1-500 delta-crystallin genes per diploid mouse genome. No expression of the exogenous genes was detected in the transformed cells as long as they were in the undifferentiated state. However, the synthesis of delta-crystallin in certain types of cells was detected immunohistologically in three lines after the differentiation. The positive cell types were unique to each line, skeletal muscle in Y delta-9, certain columnar epithelia in Y delta-2, and unidentified spindle-shaped cells in Y delta-3. Authentic delta-crystallin polypeptides with a mol. wt. of 48 000 are synthesized upon differentiation of line Y delta-3 in solid tumors in syngenic mice.
Animals↗
Steroid therapy in cardiac sarcoidosis. Increased left ventricular contractility concomitant with electrocardiographic improvement after prednisolone.
A 23-year-old woman was referred to our clinic because of pulmonary edema. The electrocardiogram on admission demonstrated eventual QS pattern in leads 1 and a VL with a poor progression of the R-wave amplitude in the precordial leads. The left ventricle was remarkably dilated with severely diminished contractility. Biopsy of the inguinal lymph nodes, liver, and myocardium confirmed the diagnosis of generalized sarcoidosis. After administration of prednisolone, the left ventricular dimension decreased, and contractility increased concomitant with an improvement of the electrocardiographic abnormalities without any aneurysm forming. We conclude that steroids may keep some jeopardized myocardium viable and that initiation of steroid therapy in an early phase does not result in formation of an aneurysm.
Adult↗
[Fundamental study of ceftriaxone in the field of obstetrics and gynecology].
Excretion of ceftriaxone (Ro 13-9904, CTRX) into female genital organs was studied by determining its concentration in the dead space exudate of the patients who received radical hysterectomy due to uterocervical cancer. Data analysis was performed by the three-compartment model. The serum peak level of CTRX in the cubital vein was 177.74 micrograms/ml 1 hour after the initiation of intravenous drip infusion for 1 hour with 1 g. The concentration of CTRX in the exudate of pelvic dead space reached a peak of 62.75 micrograms/ml 4.61 hours after the start of administration and still showed a very high level of 25 micrograms/ml at 24 hours. The AUC of the excretion into the exudate of pelvic dead space was 1,432.43 micrograms X hr/ml. These results indicate that CTRX is a clinically useful drug, being excreted into the exudate of pelvic dead space favorably.
Bacteria↗
[How animal genes are regulated in cell differentiation].
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Animals↗
Deacylation of ceramide, triacylglycerol and phospholipids in guinea pig epidermal cells.
The epidermal cells obtained by mild trypsinization of guinea pig skin were first labeled with [3H]palmitate. Interestingly, free ceramide was one of the major radioactive products. The radioactivity of ceramide was located exclusively in the N-acyl moiety, and palmitate was incorporated as such, without detectable chain modification. Sphingomyelin and glucosylceramide were not significantly labeled in spite of the active synthesis of ceramide. In order to investigate the action of lipolytic enzymes, the labeled cells were chased in a tracer-free medium. Bovine serum albumin (or fetal calf serum) and ionophore A23187 markedly stimulated the deacylation of lipids. The effects of the two stimulators were, however, distinctly different from each other. Bovine serum albumin (or fetal calf serum) caused principally the deacylation of triacylglycerol and ceramide, with little effect on that of phospholipids. In contrast, the effect of the ionophore was confined to phospholipid deacylation. In the presence of the ionophore, phosphatidylcholine and phosphatidylethanolamine were degraded to a similar extent. Palmitate was cleaved from both C-1 and C-2 positions of phosphatidylcholine. The results suggest that the lipolytic systems of the epidermal cells can respond to extracellular stimuli, and that the action of triacylglycerol lipase and ceramidase is controlled differently from that of phospholipases.
Acylation↗
Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies.
Diacylglycerol kinase (EC 2.7.1.-) was purified 1,650-fold from pig brain cytosol. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the kinase was estimated to be 78,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar value (76,000) was obtained by Sephadex G-150 gel filtration. The activity of the purified enzyme was markedly enhanced by either deoxycholate or phospholipids. The extent of activation by phospholipids was in the order of phosphatidylcholine greater than lysophosphatidylcholine greater than phosphatidylethanolamine approximately equal to phosphatidylserine greater than sphingomyelin. Other phospholipids and unsaturated fatty acids were ineffective. Phosphatidylcholines from egg yolk and pig brain, and dioleoyl phosphatidylcholine were similarly effective. Saturated phosphatidylcholines with acyl chain lengths shorter than palmitate also gave a considerable activation. The activity with phosphatidylcholine was from 1.5- to 2.5-fold higher than that measured with deoxycholate. A very small amount of phosphatidylinositol or phosphatidylglycerol potently inhibited the phosphatidylcholine-dependent (but not deoxycholate-dependent) kinase activity. The inhibition by phosphatidylinositol was varied according to its molar ratio to phosphatidylcholine. As little as about 2.5 mol per cent of phosphatidylinositol resulted in 50% inhibition of the phosphatidylcholine-dependent kinase activity. The deoxycholate- and phosphatidylcholine-dependent kinase activities showed almost the same Km values for the substrates. In both cases, the apparent Km values for ATP and diacylglycerol were 300 microM and about 60 microM, respectively. The kinase required Mg2+ for its activity. When compared to deoxycholate, phosphatidylcholine was more effective at higher Mg2+ concentrations. The deoxycholate-dependent activity showed a broad pH optimum at around 8.0, whereas the phosphatidylcholine-dependent activity formed a clear peak at pH 7.4.
Animals↗
Tissue-specific expression of a cloned chick delta-crystallin gene in mouse cells.
Crystallins are a group of soluble proteins specific to vertebrate lenses, and have been used successfully as molecular markers for studying lens differentiation. The synthesis and accumulation of delta-crystallin in the differentiating lens cells of chick, in particular, have been extensively studied (for review see refs 1-3). Recently, we have cloned a continuous stretch of a chick delta-crystallin gene which is specifically expressed in lens cells. We have injected this gene into nuclei of various mouse somatic cells. delta-Crystallin is totally absent from mammalian lenses in which it is replaced by gamma-crystallin, thus this xenogeneic injection system should allow us to study tissue-specific gene expression. We report here experiments which demonstrate that (1) the cloned delta-crystallin gene of chick is expressed in the mouse lens epithelial cells as efficiently as in the homologous chick cells; (2) delta-crystallin synthesized in the mouse lens epithelium has the native molecular weight, indicating correct splicing of the gene transcripts; (3) the expression is inefficient in the non-lens tissues examined, suggesting lens-specific expression of the delta-crystallin gene in the xenogenic environment. To our knowledge, this is the first demonstration that a cloned gene shows preferential expression in homologous cell types of different species.
Animals↗
[Experimental and clinical studies on latamoxef in the perinatal period].
Pharmacokinetics and clinical studies on the perinatal use of latamoxef (LMOX, Shiomarin), a new parenteral oxacephem antibiotic, were carried out and the results obtained were as follows: After LMOX was intravenously given to mother at a dose of 1 g, the umbilical cord serum concentration of LMOX reached to peak with 18.4 micrograms/ml in 1 hour 20 minutes and its concentration was higher than the maternal serum after 2 hours 25 minutes and decreased gradually (T 1/2 beta = 1.61 hours). The materno-fetal transfer of LMOX was 71.0%. LMOX showed the good transfer as well as other cephalosporins. After LMOX was intravenously administered to mother at a dose of 1 g, LMOX concentration in milk wasn't detectable up to 12 hours. LMOX was intravenously administered to 18 cases with premature rupture of membrane, at a daily dose of 2 g for 3--6 days. The prophylactic effects were observed in all cases, both mother and neonate. No adverse effects were observed in mother except for 1 case with low grade abnormality of S-GPT, transiently. We observed 3 neonates with jaundice (total bilirubin greater than or equal to 17.0 mg/dl) probably not related to LMOX. It is concluded that LMOX is less toxic, safe and clinically useful antibiotic for the treatment of perinatal infections.
Bacterial Infections↗
Lentoidogenesis from neural retina cells in culture is affected by interactive relationships between different cell types.
By centrifugation in a Percoll gradient, two cell fractions were separated from cell populations harvested from 8-day cultures of neutral retina cells of 3-5-day-old quail embryos. The heavy (H-) fraction contained mostly N-cells, which are considered to be putative neuronal cells, while the light (L-) fraction contained both E-cells, putative retinal glial cells, and N-cells. Determination of choline acetyltransferase activity in both fractions suggested that this enzyme is predominantly localised in N-cells. After replating the separated L-fraction for further culturing, frequent lentoidogenesis occurred from clusters of N-cells which, though few in number, were included in this fraction. Addition of H-fraction to L-fraction cells caused a significant increase in lentoidogenesis up to a ratio of N- to E-cells of 3:1. However, addition of excess H-fraction cells beyond this ratio inhibited lens differentiation. This difference in the expression of lens phenotypes resulting from the different ratios of H- and L-fraction was confirmed by monitoring the level of delta-crystallin in cultures. These results are discussed in the light of interactive relationships between N- and E-cells in the transdifferentiation of neural cells into lens in cell culture.
Animals↗
Organization of delta-crystallin genes in the chicken.
Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.
Animals↗