Structure of the 5' sequences of the human gamma-glutamyltransferase gene.

In humans, five distinct mRNAs code for gamma-glutamyltransferase (GGT). Their coding regions are identical and their 5' untranslated regions exhibit both common and type-specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5' region of the human GGT gene. The common regions of the 5' UTR are encoded by five exons, localized within a 2.4-kb region of the genomic DNA. Three of them are separated only by intron-donor or intron-acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5' UTRs in a tissue-specific manner. In addition, we have isolated a genomic fragment containing the most distal 5' sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5' RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell-type-specific manners.

An intronic promoter controls the expression of truncated human gamma-glutamyltransferase mRNAs.

We have identified and characterized a genomic DNA fragment containing the coding sequences corresponding to the human gamma-glutamyltransferase type 1 mRNA. The coding part of the gene spans over 16 kb and comprises 12 exons and 11 introns exhibiting a similar organization as for the mouse and rat GGT genes. The exons 1-7 encode the heavy subunit whereas exons 8-12 which encode the carboxy-terminal part of the heavy subunit (exon 8) and the light subunit are clustered in a 1.6-kb BglII fragment. Exons 7 and 8 are separated by a 3.9-kb intron containing in its 3' part the sequences corresponding to the 5'-UTRs of the truncated GGT mRNAs described for human lung. Sequence analysis upstream this transcribed region exhibited putative promoter sequences and after transient transfection significant promoter activities were measured in V79 lung fibroblasts and KYN-2 hepatoma cells but not in A2780 ovarian cells. This specificity disappeared when only 550 bp upstream the transcription start site were used as promoter. These results argue for a promoter of truncated GGT mRNAs in intron 7, specifically regulated in human tissues.

[Ebstein's anomaly associated with left ventricular dysfunction: a case report].

A 66-year-old man presented with Ebstein's anomaly associated with left ventricular dysfunction. He had been followed since 40 years of age for cardiomegaly and arrhythmia, and experienced episodes of orthopnea at the age of 64. He was referred to our hospital in April 1997 because of lower extremity edema. Physical examination revealed dilated external jugular vein, tenderness of the right hypocondorium, and lower extremity edema. Electrocardiography confirmed atrial fibrillation. Transthoracic echocardiography revealed bilateral atrial and ventricular dilation, and paradoxical septal movement. The apical four-chamber view demonstrated 15 mm apical displacement of the septal leaflet. Color Doppler echocardiography revealed moderate tricuspid regurgitation. Transesophageal echocardiography revealed low echoic and hypoplastic tricuspid valve. Left ventriculography showed diffuse hypokinesis, and the ejection fraction was 49%. The coronary artery was normal. Atrial septal defect was not detected. Diffuse fibrosis, which may be found in the hearts of patients with Ebstein's anomaly at autopsy may have been responsible for the left ventricular depressed systolic function in this patient.

Involvement of enhanced coagulation and fibrinolysis system in induction of atherosclerosis in hyperlipidemic rabbits fed on a high cholesterol diet.

We studied the involvement of coagulation and fibrinolysis system in the induction and development of atherosclerosis in rabbits with hyperlipidemia induced by a high-cholesterol diet (HCD). In HCD rabbits, plasma lipids and atherogenic indices were maintained at a high level throughout the experimental period compared with those in rabbits fed on a standard diet. In the early phase, a significant increase in fibrinogen level was followed by increases in the activities of plasminogen and tissue-type plasminogen activator with a decrease in alpha 2-plasmin inhibitor activity and platelet count. In the middle and late phases, significant increases in plasminogen activator inhibitor-1 and antithrombin-III were observed in HCD rabbits. These results suggest that the early enhancement of coagulation followed by high activity of fibrinolysis is involved in the induction and development of hyperlipidemic thromboembolism and atherosclerosis in HCD rabbits.

Effects of clinofibrate on plasma fibrinogen level in high fructose diet-induced hyperlipidemic rats.

Effects of clinofibrate on the coagulative and fibrinolytic activity in an intrinsic hyperlipidemia induced by a cholesterol free-high fructose diet (HFD) for 14 days were studied using male Wistar rats. There were significant and positive correlations between plasma fibrinogen level and serum lipid content, and between fibrinogen level and VLDL-LDL-lipid content. Clinofibrate administration (50 and 100 mg/kg/day, p.o.) significantly inhibited the increase in plasma fibrinogen level as well as serum- and VLDL-LDL-lipids. It is concluded that clinofibrate, in addition to its beneficial effect on the serum lipid profile, can effectively reduce the plasma fibrinogen level.

Correlation between plasma fibrinogen and serum lipids in rats with hyperlipidemia induced by cholesterol free-high fructose or high cholesterol diet.

We studied the coagulative and fibrinolytic activity in intrinsic or extrinsic hyperlipidemia using 4-week-old male Wistar rats. Intrinsic hyperlipidemia was induced by a cholesterol-free high-fructose diet (HFD) and extrinsic hyperlipidemia, by a high-cholesterol diet (HCD) for 14 days. In intrinsic hyperlipidemic rats fed on the HFD, serum lipids were significantly increased as compared with the levels in control rats fed on a standard diet. An apparent increase in plasma fibrinogen level and coagulant factor XIII activity was also observed in HFD rats. In extrinsic hyperlipidemic rats fed on the HCD, significant increases in plasma fibrinogen level compared with that of control rats were found with the increases in serum lipids. Activities of antithrombin III and alpha 2-plasmin inhibitor in HFD-fed rats significantly increased compared with those of control and HFD rats. There was a significant positive correlation between plasma fibrinogen and serum total cholesterol, free cholesterol, or phospholipid in diet-induced hyperlipidemia (p < 0.01). Because of the increase in coagulant XIII activity in HFD-fed rats and the increase in alpha 2-plasmin inhibitor activity in HCD-fed rats, both diet-induced hyperlipidemic rats were shown to have enhanced coagulative activity compared with the control rats. These results suggest that the HFD as well as the HCD causes a pre-hypercoagulative state due to the increase in plasma fibrinogen level and activities in other coagulative and fibrinolytic factors.

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis.

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V ps andV px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO2. Prolonged exposure of the hypocotyl to high concentrations of CO2 (above 10%) caused depolarization of membrane potentials above the level before CO2 introduction. When CO2 was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO2 as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO2-induced maximum hyperpolarization ofV ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO2 may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.