Molecular cloning of the gene coding for the human T cell differentiation antigen CD7

The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites — 122 base pairs (bp) to — 38 bp from ATG translation initiation site were demonstrated. The promoter region had high G+C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

Adenosine deaminase activity in relation to the appearance of early and late Epstein-Barr virus antigens induced in lymphoblastoid cells

Summary Induction of Epstein-Barr virus (EBV) capsid antigen synthesis in 59.6% of P3HR-1 cells was followed by a decrease to 70% in adenosine deaminase (ADA) activity. In Daudi cells synthesizing EBV early antigen, ADA activity did not decrease.     

Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis.

BmFTZ-F1 is a Bombyx mori homologue of FTZ-F1, a positive regulator of the fushi tarazu gene of Drosophila melanogaster. In order to determine the sequence recognized with this factor, we made three sets of oligonucleotide mixture which contain 4 possible nucleotides at different positions within the previously proposed 12-bp binding consensus sequence. Oligonucleotides which bound to purified BmFTZ-F1 were separated by a gel mobility shift procedure and a binding sequence was determined by direct sequencing through Maxam-Gilbert method. By this analysis, 7 positions showed clear sequence preference and 5 positions showed weak or no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition analysis and results were presented as a quantitative difference in the binding affinity. From these analyses, we conclude that the best binding sequence of BmFTZ-F1 is 5'-PyCAAGGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.     

A novel cloverleaf structure found in mammalian mitochondrial tRNA(Ser) (UCN).

Bovine mitochondrial tRNA(Ser) (UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated the bovine liver mitochondrial tRNA(Ser) (UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5'-terminal nucleotide and enzymatic determination of the whole sequence of tRNA(Ser) (UCN) revealed that the tRNA started from the third nucleotide of the putative tRNA(Ser) (UCN) gene, which had formerly been supposed. Enzymatic probing of tRNA(Ser) (UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNA(Ser) (UCN) genes of mammals such as human, rat and mouse, but not for those of non-mammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria.     

Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils

The rate of superoxide generation of guinea pig intraperitoneal neutrophils by a chemotactic peptide or 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased by 2-bromo-2-chloro-1,1,1,-trifluoroethane (halothane), an inhalation anesthetic. This increase was inhibited by 1-(5-isoquinolinesulfonyl)methylpiperazine dihydrochloride (H-7), a specific inhibitor of Ca2+- and phospholipid-dependent protein kinase C (PKC). Halothane was found to significantly activate partially purified PKC. The activation required phosphatidylserine (PS) and Ca2+. Dioleoylglycerol- or TPA-activated PKC activity was further increased by halothane. The cytoplasmic proteins of guinea pig neutrophils phosphorylated by halothane-activated PKC were similar to those phosphorylated by PMA-activated PKC. The phosphorylation of a 48 kDa protein, a phosphorylated protein required for NADPH oxidase activation, was also increased by halothane. These data suggest that the increase of superoxide production by halothane is correlated with its activation of PKC.     

Identification of both calpains I and II in nucleated chicken erythrocytes

Chicken erythrocytes were found to contain two species of calpains which differ in elution profile from DEAE-cellulose and in Ca2+ requirement. After partial purification, one of them was half-maximally activated by 10 microM Ca2+ and the other by 180 microM Ca2+. The low- and high-Ca2+-requiring proteases cross-reacted only with the respective monospecific antibodies for mammalian calpain I and calpain II, respectively. Approximately 5 times more calpain I than calpain II is present in chicken erythrocytes. By immunoelectrophoretic blot analysis, both calpains I and II from chicken erythrocytes were proved to be heterodimers composed of 76 and 28 kDa, and 80 and 28 kDa subunits, respectively. Our present finding that the heavy subunit of calpain I is smaller than that of calpain II is noteworthy, since the opposite is known to be true of various mammalian calpains. An immunological study has revealed that the calpain I newly found in chicken erythrocytes is not derived from calpain II. Thus, the co-existence of calpains I and II in one animal species also holds in chickens, contrary to the previously advocated notion that chickens have only one type of calpain.     

Changes in aerobic and anaerobic ATP-synthesizing activities in hypoxic mouse brain

The changes in cerebral metabolism in mice in severe hypoxia were investigated by analyses of changes in the levels of energy metabolites and near-infrared spectrophotometric assessment of the states of hemoglobin and cytochrome oxidase. Under 4.4% O2, the contribution of anaerobic ATP production was at most about 20% of the demand. However, the cerebral ATP level was kept at the control level until about 1 min before death. Pentobarbital anesthesia, which reduced the cerebral rate of metabolism, prolonged the survival time, although anaerobic ATP production still did not support ATP demand. Under these conditions, deoxygenation of hemoglobin and reduction of cytochrome oxidase proceeded rapidly within 1 min. Hemoglobin reached the maximum state of deoxygenation in the middle phase of hypoxia, with no further change until death. However, cytochrome oxidase was reduced slowly with one phase of partial reoxidation due to increase of cerebral blood volume, and reached the completely reduced state at death. From these results it was concluded that the aerobic ATP synthesis, which supplied more than 80% of the cerebral demand, decreased gradually because of limitation of oxygen supply, and that the failure of oxidative phosphorylation to meet demand triggered the decrease in the cellular ATP level that led to death.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.