Relationships between cell surface protease and acid phosphatase activities ofLeishmania promastigote

A correlation between the ratio of the cell surface protease activity to phosphatase activity and the complexity of the pattern of cell surface exposed polypeptides ofLeishmania promastigotes was demonstrated for various strains grown under similar conditions. The ratio of the cell surface protease activity to acid phosphatase activity was high forL. major andL.b. panamensis and it correlates with the expression of a single polypeptide of 63 KDa on their cell surface. Intermediate and lower ratios of these enzymatic activites relate with more complex radio-iodinated patterns: two main bands inL.b. guyanensis (70 and 58 KDa) andL.b. braziliensis (72 and 60 KDa) and three main bands 65, 50, 27 KDa in allL.m. mexicana strains tested. Evidence is presented that the acid phosphatase located on theL.m. mexicana cell surface is not an artifact due to a secondary absorption of the secreted acid phosphatase from the culture medium. These results confirm theLeishmania antigen cell surface heterogeneity. The implications on the biology ofLeishmania and the clinical manifestation of leishmaniasis are discussed.     

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

Crystallization and preliminary X-ray analysis of nonglycosylated tissue inhibitor of metalloproteinases-1, N30QN78Q TIMP-1

A nonglycosylated (N₃₀QN₇₈Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2₁, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.     

FLORAL INITIATION AND EARLY DEVELOPMENT IN ERIGERON PHILADELPHICUS (ASTERACEAE)

The order of floral initiation and subsequent organogeny of Erigeron philadelphicus L. (Asteraceae: Astereae) was found to deviate from the acropetal pattern generally reported for the Asteraceae. Light micrographs show periclinal divisions in the first, second, and deeper subsurface layers of cells on the flanks of the inflorescence apex as the earliest evidence of floral initiation. Scanning electron microscope micrographs indicate that the disk flowers appear first and arise as small protuberances approximately one-third of the way up the previously and undifferentiated highly convex inflorescence apex. A succession of disk flowers arises acropetally in a complex anthotaxy characterized by about 21 dextrorse and 12–15 sinistrorse parastichies (although this pattern is obscured at the apex). After one to three disk flowers have been initiated in each parastichy, the first ray flower initials can be seen to initiate in sites proximal to the oldest and largest disk flowers. Additional ray flowers then initiate basipetally following the dextrorse parastichies established by the disk flowers. Overall floral initiation on the inflorescence apex proceeds acropetally for the disk flowers and basipetally for the ray flowers until the available space is filled. Floral development adheres to the same plan—proceeding bidirectionally on the inflorescence meristem with the oldest and most complete flowers of both types located on the equator established at initiation.     

Nicardipine inhibits axon conduction but causes dual changes of acetylcholine release in the mouse motor nerve

The effects of nicardipine, a dihydropyridine Ca2(+)-channel antagonist, on neuromuscular transmission and impulse-evoked release of acetylcholine were compared with those of nifedipine. In the isolated mouse phrenic nerve diaphragm, nicardipine (50 microM), but not nifedipine (100 microM), induced neuromuscular block, fade of tetanic contraction, and dropout or all-or-none block of end-plate potentials. Nicardipine had no significant effect on the resting membrane potential and the amplitude of miniature end-plate potentials but increased the frequency and caused the appearance of large size miniature potentials. The quantal contents of evoked end-plate potentials were increased. In the presence of tubocurarine, however, nicardipine depressed the amplitude of end-plate potentials. The compound nerve action potential was also decreased. It is concluded that nicardipine blocks neuromuscular transmission by acting on Na+ channels and inhibits axonal conduction. Nicardipine appeared to affect the evoked release of acetylcholine by dual mechanisms, i.e., an enhancement presumably by an agonist action on Ca2+ channels, like Bay K 8644 and nifedipine, and inhibition by an effect on Na+ channels, like verapamil and diltiazem. In contrast with its inactivity on the amplitude of miniature end-plate potentials, depolarization of the end plate in response to succinylcholine was greatly depressed. The contractile response of baby chick biventer cervicis muscle to exogenous acetylcholine was noncompetitively antagonized by nicardipine (10 microM), but was unaffected by nifedipine (30 microM). These results may implicate that nicardipine blocks the postsynaptic acetylcholine receptor channel by enhancing receptor desensitization or by a use-dependent effect.     

Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading Acinetobacter Strain

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.     

Detection of truncated virus particles in a persistent RNA virus infection in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics of trout and salmon fry in hatcheries around the world. In laboratory and field studies, epizootic survivors are negative for infectious virus by plaque assay at about 50 days postexposure. Survivors are considered virus free with no sequelae and, thus, are subsequently released into the wild. When adults return to spawn, infectious virus can again be isolated. Two hypotheses have been proposed to account for the source of virus in these adults. One hypothesis contends that virus in the epizootic survivors is cleared and that the adults are reinfected with IHNV from a secondary source during their migration upstream. The second hypothesis contends that IHNV persists in a subclinical or latent form and the virus is reactivated during the stress of spawning. Numerous studies have been carried out to test these hypotheses and, after 20 years, questions still remain regarding the maintenance of IHNV in salmonid fish populations. In the study reported here, IHNV-specific lesions in the hematopoietic tissues of rainbow trout survivors, reared in specific-pathogen-free water, were detected 1 year after the epizootic. The fish did not produce infectious virus. The presence of viral protein detected by immunohistochemistry, in viral RNA by PCR amplification, and in IHNV-truncated particles by immunogold electron microscopy confirmed the presence of IHNV in the survivors and provided the first evidence for subclinical persistence of virus in the tissues of IHNV survivors.     

Modulation of IL-4 production in murine spleen cells by prostaglandins

Recently, it has been reported that IL-4 production by murine Th2 cell lines is insensitive to inhibition by E-type prostaglandins. In the present study, IL-4 production in vitro by freshly isolated concanavalin A (Con A)-stimulated murine spleen cells was readily suppressed by PGE₂ with an I₅₀ of 2 nM. Comparable suppression by PGE₂ was seen after priming by anti-CD3? antibody instead of Con A or with other changes in the culture conditions. PGE₂ was an effective inhibitor after elimination of Ly2.2⁺ T cells, consistent with a direct effect on Th2 cells. In the absence of added prostaglandins, IL-4 production was enhanced 1.5- to 7.0-fold by 0.2–2.0 μM indomethacin, indicating that endogenous arachidonate metabolites such as PGE₂ and PGI₂ regulate IL-4 production in our usual culture system. The inhibition of Th2 cell secretion by PGE₂in vitro may have physiologic and pharmacologic implications for the regulation of Th2 cell function and IgE production in vivo.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.