Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Asexual reproductive organ-specific expression of the glyceraldehyde-3-phosphate dehydrogenase 2 gene of Pilobolus crystallinus

Pilobolus crystallinus has three putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes (pcgapdh1, pcgapdh2 and pcgapdh3). The results of this study demonstrate that expression of pcgapdh2 was increased by irradiation and that this increased expression was correlated with the formation of asexual reproductive organs (trophocysts). Interestingly, expression of pcgapdh2 was restricted to trophocysts. The formation of trophocysts was likely promoted by light, and the expression of pcgapdh2 was increased as a result of trophocyst formation. This is the first report that shows the regulation of a gapdh gene in an organ-specific manner in fungi.     

Disappearance of terminal deoxynucleotidyltransferase in human malignant T-lymphoblasts treated with a human alpha interferon preparation

Human T-lymphoblastoid cell lines RPMI 8402, MOLT-3, and CCRF-CEM were treated with interferon (IFN) to determine if the treatment would result in the disappearance of cellular terminaldeoxynucleotidyltransferase (TdT), a possible differentiation marker for T-lymphocytes. Incubation of RPMI 8402 cells in the presence of IFN preparation caused a decrease in the number of TdT-positive cells and in TdT activity of the cell extract. The inhibition of cell multiplication was dose dependent. The anticellular effect of IFN preparation was cytostatic, not cytocidal. The IFN preparation modified neither the TdT content nor proliferation of MOLT-3 and CCRF-CEM cell lines. The effects of IFN preparation thus varied with the cell line.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

Modulation of brain progestin and glucocorticoid receptors by unsaturated fatty acid and phospholipid

In an attempt to learn how nonsteroidal factors modulate brain progestin and glucocorticoid receptors, the effects of saturated and unsaturated fatty acids, and phosphatidylinositol on the binding of [3H]R5020 or [3H]dexamethasone, determined by sucrose density gradient and gel filtration on LH20, were examined in the cerebral cortical cytosol from 10-day-old female rats which contain a considerable amount of progestin and glucocorticoid receptors. Unsaturated fatty acids such as oleic (C18:1), arachidonic (C20:4) and docosahexaenoic acid (C22:4) depressed the [3H]R5020 or [3H]dexamethasone binding in increasing order, but saturated fatty acids had no effect. Arachidonic and docosahexaenoic acids, which were strong inhibitors, lowered the binding dose dependently. The fatty acid inhibition on brain progestin and glucocorticoid receptors was thus a function of acid dose and degree of acid unsaturation. Interestingly, prostaglandin D2 did not show any effect. Among phospholipids tested the inhibitory effect of phosphatidylinositol on the [3H]R5020 binding was evident, but no significant effect was found with phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine or sphingomyelin. The phosphatidylinositol inhibition was dose dependent. Analysis on kinetics and Scatchard plot have revealed the noncompetitive type of inhibition by arachidonic acid and phosphatidylinositol. From these results it is suggested that the unsaturated nonestrified fatty acid, arachidonic acid, and phosphoinositides modulate the brain progestin and, possibly, glucocorticoid receptors through their binding at sites different from steroid binding sites on the respective receptor molecules.     

Infrageneric variation of the low molecular weight carbohydrate composition of the seeds of the genusVicia (Leguminosae)

The low molecular weight carbohydrate compositions of the seeds of 29 species ofVicia, namelyV. amoena, V. amurensis, V. bifolia, V. dumetorum, V. fauriei, V. japonica, V. nipponica, V. pisiformis, V. pseudo-orobus, V. sylvatica, V. unijuga, V. venosa, V. cassubica, V. orobus, V. cracca agg.,V. hirsuta, V. villosa agg.,V. tetrasperma,V. oroboides, V. sepium, V. cuspidata, V. grandiflora, V. lathyloides, V. sativa agg.,V. bithynica, V. faba, V. narbonensis, V. hybrida andV. lutea were determined by gas liquid chromatography. The carbohydrate compositions were found to be species-specific. Principal component analysis of the carbohydrate composition data showed that these species can be divided into three groups. Although, as far as the examined species were concerned, these groups were not correlated with the known subgenera, significant correlation between the groups and the known sections was detected in the subgenusVicia. The carbohydrate composition character would be important to clarify the relationships among closely related taxa of the genusVicia.     

Multiple forms of calpastatin in pig brain

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.     

Development of nephrotic ICGN mice--the origin, reproductive ability, and incidence of glomerulonephritis

ICGN is a strain of spontaneous nephrotic mice with nonproliferative glomerular lesions. It was derived from an outbred Yok: ICR colony in our laboratory. The renal disease constantly occurred in animals of the first to the tenth generations (greater than 13.0%; 70 days of age). When affected males were mated with unaffected females, the incidence of the disease in their offspring was 38.8% (n = 49) at 70 days after birth. When both parents were affected, their offspring were all affected (n = 12). The disease evenly progressed in both sexes. It usually began 40 to 150 days after birth and death occurred within two months after onset. The animals usually showed sufficient reproductive ability as long as unaffected females were used for mating.     

The direct activation of human multidrug resistance gene (MDR1) by anticancer agents

Enhanced expression of a multidrug-resistance gene (MDR1) is observed in some cancer patient, but any regulatory mechanisms of MDR1 gene expression in this phenomenon is not yet known. In this study, the regulation of MDR1 gene was analysed by transient expression assays in the presence of anticancer agents. We found that MDR1 promoter could be activated directly on the addition of anticancer agents including vincristine, daunomycin, adriamycin and colchicine. The results suggest that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype.