Detection by ELISA of Two Tobamoviruses in Orchids Using Monoclonal Antibodies

Five monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus-coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS-ELISA. None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal-monoclonal antibody DAS-ELISA system is advocated for detecting both tobamoviruses in orchids.     

Diurnal variation in 5′-nucleotidase activity in rat liver

Summary The diurnal variation of 5-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora.

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.     

Inhibition of human neutrophil collagenase by gold(I) salts used in chrysotherapy

Six gold(I) salts, some of which are used as drugs in chrysotherapy, are shown to be inhibitors of two forms of human neutrophil collagenase. The IC50 values vary over six orders of magnitude, the lowest being 3.5 nM for Myocrisin. Thus, inhibition is greatly affected by the identity of the ligands to the gold(I) atom. The inhibition of collagenase by these gold(I) salts may be a partial basis for their antiarthritic action.     

Prevalence of polymorphic 21-hydroxylase gene (CA21HB) mutations in salt-losing congenital adrenal hyperplasia

Using genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, we identified three different CA21HB mutation patterns: no detectable restriction fragment abnormalities (16/28 haplotypes), deletion of the active CA21HB gene (9/28), and apparent conversion of the active CA21HB gene to the pseudogene CA21HA (3/28). CA21HB gene deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7. A variety of HLA and C4 types was associated with the other mutations. Apparent conversion of CA21HB to CA21HA was identified by the disparity between the intensity ratios for the major TaqI and BglII hybridization fragments.     

Kinetics and concentration dependency of cAMP-induced desensitization of a subpopulation of surface cAMP receptors in Dictyostelium discoideum

Extracellular cAMP induces the rapid activation of guanylate cyclase, which adapts within 10 s to constant cAMP concentrations. A new response can be induced either by a higher cAMP concentration or by the same cAMP concentration at some time (t1/2 = 90 s) after removal of the previous stimulus. Stimulation of guanylate cyclase is supposed to be mediated by a subpopulation of cell surface cAMP receptors (B-sites). These sites can exist in three states, BF, BS, and BSS, which interconvert in a cAMP and guanine nucleotide dependent manner. It has been proposed that the transition of BS to BSS represents the activation of a guanine nucleotide regulatory protein [Van Haastert, P.J.M., De Wit, R.J.W., Janssens, P.M.W., Kesbeke, F., & DeGoede, J. (1986) J. Biol. Chem. 261, 9604-9611]. Binding of [3H]cAMP to these sites was measured after a short preincubation with an identical concentration of nonradioactive cAMP. [3H]cAMP could still bind to BF and BS, but not to BSS, indicating that the transition of BS to BSS is blocked by the preincubation with cAMP. This blockade was rapid and showed first-order kinetics with t1/2 = 4 s. A half-maximal blockade was induced by 0.7 nM cAMP; at this concentration only 5% of the B-sites are occupied with cAMP. The blockade of the transition of BS to BSS was released by two conditions: (i) When the concentration of cAMP was increased, the blockade was released within a few seconds. (ii) When cAMP was removed, the blockade was released slowly with t1/2 = 90 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of amino acid transport system B0,+ in mouse blastocysts

The capacity of preimplantation mouse blastocysts to express the novel amino acid transport activity provisionally designated system B0,+ increased approximately 3-fold 1 day after administration of estrogen to their progesterone-primed, ovariectomized mothers. Nevertheless, blastocysts obtained 22-25 h after estrogen administration (implanting blastocysts) had to be incubated in vitro for about 20 min before they fully expressed their B0,+ activity. No similar increase in B0,+ activity was observed upon incubation of blastocysts obtained before estrogen administration (diapausing blastocysts). Rapid metabolic changes can be induced in the uterus by massaging it with a blunt instrument while it is receptive to implantation, and this treatment was found to increase the apparent B0,+ activity in implanting but not diapausing blastocysts. In contrast, the activity of an incompletely characterized, Na+-independent system, which accepts L-lysine as a substrate, decreased more than 2-fold when implanting blastocysts were incubated in vitro. No change in Na+-independent lysine uptake was detected during incubation of diapausing blastocysts. It is suggested that both uteri and blastocysts develop the capacity to change rapidly some of their metabolic processes near the time of implantation, and one of the processes which may be subject to rapid change in blastocysts is amino acid transport. These developmental events appear to coincide with and could be required for the decidual cell response and implantation of blastocysts in the uterus.     

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.     

Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri

Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion. Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70%. Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion). In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored. This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion). Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation.