Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins.

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.     

Rapid detection and prevalence of cholesteryl ester transfer protein deficiency caused by an intron 14 splicing defect in hyperalphalipoproteinemia

A deficiency of plasma cholesteryl ester transfer protein (CETP) is one of the genetic causes of increased serum high density lipoprotein (HDL)-cholesterol levels (hyperalphalipoproteinemia). A splicing defect (GA mutation) at the +1 position of intron 14 of the human CETP gene is a common mutation in the Japanese CETP deficiency. A rapid screening method for the splicing defect by means of primer-specified restriction map modification was described. The frequency of the mutation in hyperalphalipoproteinemia was determined, and its frequency in the general population was estimated. During polymerase chain reaction (PCR) with a modified primer, a novel NdeI restiction endonuclease site was created from the mutated allele in the PCR products, which could be visualized after electrophoresis of the digested products. As a result, 21 of 121 unrelated hyperalphalipoproteinemic subjects with HDL-cholesterol 60 mg/dl (1.55 mmol/1), were found to havethe GA mutation. Of the 21 individuals, 8 were found to be homozygous for the mutation. Allele frequency of the mutation was 1.5% (1/68), 2.8% (2/72), 7.1% (4/56), and 47.8% (22/46) in the groups with HDL-cholesterol levels of 60–79 mg/dl, 80–99 mg/dl, 100–119 mg/dl, and 120 mg/dl, respectively. Based on the percentage of the area under the computed normal distribution curve of serum HDL-cholesterol, the frequency of the mutated allele in the general population was estimated to be 0.81 % from the present results. This rapid detection method facilitates large-scale screening of CETP deficiency caused by the splicing defect. The mutation was frequent in Japanese subjects with hyperalphalipoproteinemia, especially in the group with HDL-cholesterol 120 mg/dl.     

Primary defect of juvenile visceral steatosis (jvs) mouse with systemic carnitine deficiency is probably in renal carnitine transport system

We investigated the reabsorptional system for carnitine in the kidney to elucidate the mechanism of carnitine deficiency in juvenile visceral steatosis (jvs) mice. Jvs mice had a higher rate of carnitine excretion at 10 days after birth than the controls, in spite of having no pathological acylcarnitine in the urine. In an experiment to assay the uptake of carnitine using kidney slices, homozygous mutants showed significantly lower rates of Na-dependent carnitine uptake than controls. Heterozygous mice showed values of transport activity intermediate between homozygous mutants and homozygous controls. Scatchard plots (transport activity versus transport activity/carnitine concentration) revealed that the homozygous mutants had a defect in the hihg affinity site (K_m = 58 μM) in the Na-dependent carnitine transport system in the kidney. These results indicate that the primary defect of jvs mice is most probably related to the system for reabsorption of carnitine in the kidney.     

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes

Exposure of rat hepatocytes to cadmium below 50 μM for a short period (10 min) resulted in cellular acidification. Conversely, exposure to Cd more than 50 μM for a long period (60 min) caused cellular alkalinization accompanied by membrane damage as reflected by decrease in cellular K content and loss of intracellular lactic dehydrogenase. In hepatocytes exposed to 5 μM Cd, a concentration sufficient to induce acidification without cytotoxicity, the metal was preferentially associated with the crude nuclei and cell debris fractions, suggesting an interaction between Cd and cell membranes to cause acidification. Omission of bicarbonate from the incubation medium induced cellular acidification. The presence of Cd in this medium did not potentiate the medium-induced acidification. Mg-ATP (25 μM) induced cellular acidification in relation to an increase in the concentration of cytosolic free Ca. The coexistence of Mg-ATP and Cd at the concentrations which had no effect on cellular pH in the presence of either agants induced cellular acidification. These observations suggest that Cd induced cellular acidification by modulating the process connected with the rise in cytosolic free Ca via interaction with plasma membranes. This acidification had no strong immediate cytotoxic actions but led to subsequent cellular alkalinization accompanied with severe cytotoxicity and membrane breakage.     

Jasmonate-inducible expression of a potato cathepsin D inhibitor-GUS gene fusion in tobacco cells

A potato gene encoding cathepsin D inhibitor (CDI) is expressed constitutively in tubers and flower buds and it is inducible in leaves upon wounding of the tissue or by treatment with methyl jasmonate (MJA). A fusion gene (CDI:GUS) in which the 2.4 kb long promoter of the CDI gene was translationaly fused with the coding sequence for -glucuronidase (GUS) showed MJA-inducible expression in transformed tobacco cells in suspension. The maximum level of induction by MJA was obtained in the absence of auxin and repression of MJA-inducible expression of the fusion gene by auxin was released by aphidicolin, the results suggesting that MJA-inducible expression is repressed during active cell division. JA and MJA showed similar activities in inducing the expression of the fusion gene, while other JA-related compounds such as cucurbic acid, tuberonic acid and dihydrojasmonic acid neither induced expression of the fusion gene nor inhibited the MJA-inducible expression of the fusion gene. Methyl dihydrojasmonate specifically stimulated the MJA-inducible expression of the fusion gene. The MJA-inducible expression of the fusion gene was observed even with a 100 bp long promoter of the CDI gene albeit with significantly decreased level of expression compared to the 2.4 kb long promoter. The 100 bp long CDI promoter did not contain a G-box or hexamer motif that had been implicated in the MJA-responsive expression of several other plant genes. Further mutagenesis of the 100 bp long promoter by deletion or oligonucleotide insertion suggested that although a sequence between –100 and –82 is required for the MJA-responsive expression, the presence of this sequence alone does not confer the MJA-responsive expression.Abbreviations BA N⁶-benzyladenine - CA cucurbic acid - CaMV cauliflower mosaic virus - CDI cathepsin D inhibitor - DMSO dimethyl sulfoxide - GUS -glucuronidase - HJA dihydrojasmonic acid - JA Jasmonic acid - MCA methyl cucurbate - MJA methyl jasmonate - MHJA methyl dihydrojasmonate - MTA methyl tuberonate - PI-II proteinase inhibitor II - TA tuberonic acid - 2,4-D 2,4-dichlorophenoxyacetic acid.     

Scanning electron microscopy of the muscle system of Hydra magnipapillata

The fine structure of the ectodermal and endodermal muscle layers of Hydra magnipapillata has been analyzed by scanning electron microscopy after hydrolytic removal of the mesoglea with NaOH and subsequent exposure of the basal and lateral aspects of the layers by mechanical dissection. The ectodermal muscle layer consists of fibrous processes of epithelial cells extending longitudinally to the body axis, whereas the endodermal muscle layer comprises cells with hexagonal bases and several strands of myonemes oriented circularly. In each layer, the muscular elements tightly interdigitate, extending a continuous muscle sheet along the mesoglea. The ectodermal and endodermal muscle sheets communicate with each other via foliate microprojections penetrating the mesoglea. On the lateral aspect of the ectodermal epithelium, spiny nerve fibers run along the upper surface of the muscle processes. The spines are often attached to muscle processes, suggesting that the former monitor muscle contraction. Nerve fibers occasionally come into contact with the mesoglea through narrow gaps between the muscle processes. In the hypostomal ectoderm, a small spindle-shaped cell, probably sensory in nature, extends an apical cilium and a long basal process.     

Pulmonary vasoconstriction induced by mitral valve obstruction in sheep

We hypothesizedthat left atrial hypertension results in pulmonary vasoconstriction,which is obscured by the expected passive decrease in pulmonaryvascular resistance. The objectives of this study were todemonstrate and quantify the vasoconstrictive changes that occur in thepulmonary circulation during experimental left atrial hypertension, todetermine the site of vasoconstriction, and to explore its mechanism.Sheep were instrumented for measurement of pulmonary arterial (Ppa),left atrial (Pla), and systemic arterial pressures (Psa) with a Foleyballoon catheter to variably obstruct the mitral valve. Distalpulmonary arterial wedge pressure (Ppaw) was determined by using a 5-FrSwan-Ganz catheter that was advanced until it wedged with the balloondeflated. Cardiac output (CO) was estimated by thermodilution;pulmonary vascular resistances (PVR) were calculated as mean (Ppa  Pla)/CO = total PVR, (Ppa  Ppaw)/CO = upstream PVR, and(Ppaw  Pla)/CO = downstream PVR. We studied 15 awake sheep atbaseline and during increases in Pla of 10 and 20 cmH₂O, with and without inhalationof ~36 parts per million of nitric oxide. Left atrial hypertensionresulted in elevation of Ppa. CO decreased only slightly at both levels of Pla elevation. Nitric oxide inhalation caused a significant decreasein PVR, which was greater as Pla increased. This vasodilator effect wasmost striking in downstream vessels. Experiments with phentolamine,atropine, and ibuprofen failed to reveal the mechanism of the reactivepulmonary vasoconstriction.

     

Epstein-Barr Virus Contributes to the Malignant Phenotype and to Apoptosis Resistance in Burkitt’s Lymphoma Cell Line Akata

In the present study, we established an in vitro system representing the Burkitt’s lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

INDUCTION OF HEPATOCYTE GROWTH FACTOR IN THE LIVER, KIDNEY AND LUNG FOLLOWING TOTAL BODY IRRADIATION IN RAT

Hepatocyte factor (HGF) has been shown to have a pleiotropic function to act as a potent organotropic factor in the regeneration of injury in various organs, including the liver, kidney and lung. To examine the involvement of HGF in radiation injury, the authors analysed the changes in HGF mRNA and HGF protein levels in the rat organs (liver, lung, kidney) and plasma following 6 Gy of total body irradiation. Expression of HGF mRNA in the liver and kidney increased 6–48 h after total body irradiation and returned to previous values 1 week later. HGF protein levels in lung and liver showed 1.3-2 fold elevations 1–2 weeks after irradiation (P< 0.05). HGF levels in plasma stayed at undetectable levels up to 1 month after total body irradiation. The labelling index determined 2 weeks and 1 month after total body irradiation indicated no enhancement of regeneration. Thus, total body irradiation induced transient HGF elevation in these organs without enhancement of regeneration