Evidence from two-protonic-state reactivity probe kinetics that chymopapain in fresh nonfruit latex ofCarica papaya consists of multiple forms of chymopapain A. The value of catalytic site characteristics in the identification,classification, and characterization of the papaya cysteine proteinases papain,the chymopapains,and papaya proteinase Ω

Fresh latex ofCarica papaya was collected from the stem, leaves, and petioles of the growing plant and fractionated by ion-exchange chromatography on a column of SP-Sephadex-C50 and by FPLC using a Mono S column. The fractions were examined for catalytic activity using Z-Lys-ONp andl-BAPNA as substrates and the thiol contents and reactivity characteristics were determined by using 2,2′-dipyridyl disulfide as a two-protonic-state thiol titrant and reactivity probe. By these methods the fresh nonfruit latex was shown to contain papain (EC 3.4.22.2), multiple forms of chymopapain, all of which have catalytic site reactivities characteristic of chymopapain A, and papaya proteinase Ω (originally called papaya peptidase A). The necessity now to characterize the catalytic site of a chymopapain in order to identify it is discussed.     

Brain adenosine 5′-triphosphate–creatine phosphotransferase. Purification, thiol group reactivity and the amino acid sequence around the reactive thiol groups

1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.     

Isolation and characterization of β-d-glucan,heteropolysaccharide, and trehalose components of the basidiomycetous lichen Cora pavonia

Cora pavonia, a lichen having a basidiomycetous mycobiont, is rich in protein (36%), of which 10% is tyrosine. α,α-Trehalose is present and was isolated in 4.4% yield. The lichen was found to contain polysaccharides typical of basidiomycetes and different from those of ascomycetes and ascomycetous lichens. Isolated and characterized were a β-d-glucan and a heteropolysaccharide containing l-rhamnose, l-fucose, d-xylose, d-mannose, d-glucose, and d-galactose. The β-d-glucan was highly branched with 21% of nonreducing end-groups, contained 3-O-, 6-O-, and 3,6-di-O-substituted β-d-glucopyranosyl units, and had a main chain consisting of (1→3)- and (1→6)-links. The heteropolysaccharide component contained mainly mannose and xylose, having a mannose-containing nucleus and a main chain with preponderant (1→3)-linked α-d-mannopyranosyl residues. These were unsubstituted (10%), and 4-O- (10%) and 2,4-di-O-substituted (10%) with residues of β-d-xylopyranose. On methylation analysis of the heteropolysaccharide, a capillary column of DB-210 proved to be particularly useful for gas-liquid chromatographic resolution of partially O-methylated alditol acetates.     

Isolation and characterization of CcAbf62A, a GH62 α-L-arabinofuranosidase, from the basidiomycete Coprinopsis cinerea

A cDNA encoding a putative extracellular α-L-arabinofuranosidase was cloned from the basidiomycete Coprinopsis cinerea (CcAbf62A). CcAbf62A belongs to glycoside hydrolase family 62 (GH62), but is phylogenetically distinct from previously characterized GH62 enzymes. The recombinant CcAbf62A, expressed in Pichia pastoris, released L-arabinose from both wheat arabinoxylan and oat-spelt xylan. The enzyme activity for wheat arabinoxylan was increased by the addition of CcEst1, a carbohydrate esterase from C. cinerea.     

Isolation and characterization of cDNAs encoding PDE5A,a human cGMP-binding,cGMP-specific 3′,5′-cyclic nucleotide phosphodiesterase

Human cGMP-binding, cGMP-specific 3′,5′-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100 012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5′-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25–27.     

The effect of intravital chelation with dimercaprol,calcium disodium edetate, 1-10-phenantroline and 2,2′-dipyridyl on the sulfide silver stainability of the rat brain

Summary Dimercaprol, calcium disodium edetate, 1-10-phenantroline and 2,2-dipyridyl were injected intraperitoneally in adult albino rats in doses ranging from 10 to 1500 mg per kg body weight. Ten animals were injected intracerebrally. At various survival times (2 minutes to several hours) their effects on the staining pattern of heavy metals as revealed by the sulfide silver method of Timm were determined. EDTA was virtually ineffective while dimercaprol, phenantroline and dipyridyl reduced the staining in many regions of the brain. A noteworthy effect of phenantroline was that it reduced the staining of a cortical area, corresponding remarkably well in extent, shape and location to the neocortical somatosensory area.     

Phosphate carrier of liver mitochondria: The reaction of its SH groups with mersalyl, 5,5′-dithio-bis-nitrobenzoate,andN-ethylmaleimide and the modulation of reactivity by the energy state of the mitochondria

The inhibitory effect of three SH reagents, mersalyl, 5,5-dithio-bis-nitrobenzoate, andN-ethylmaleimide, on P_i transport in rat liver mitochondria was investigated under a variety of conditions. Mersalyl binds at room temperature with both high (K _d<10 µM) and low affinity to mitochondria. Inhibition of P_i transport by mersalyl goes in parallel with titration of the high-affinity sites, inhibition being complete when 3.5–4.5 nmol/mg protein is bound to the mitochondria. At concentrations of mersalyl equal to or higher than 10 µM, inhibition of P_i transport occurs in less than 10 sec. At concentrations of mersalyl lower than 10 µM, the rate of reaction with the P_i carrier is considerably decreased. At a concentration of 100 µM, 5,5-dithio-bisnitrobenzoate fully inhibits P_i transport in about 1 min at room temperature. Nearly total inhibition is attained when as little as 40–50 pmol/mg is bound to mitochondria. Upon incubation longer than 1 min, additional SH groups, not belonging to the P_i carrier, begin to react. The uncoupler carbonyl cyanidep-trifluoromethoxyphenylhydrazone decreases the rate of reaction of mersalyl, 5,5-dithio-bis-nitrobenzoate, andN-ethylmaleimide with the P_i carrier. Preincubation with P_i has a similar effect. We propose that both carbonyl cyanidep-trifluoromethoxyphenylhydrazone and P_i act by increasing the acidity of the mitochondrial matrix. Protonation of the P_i carrier at the matrix side would change the accessibility of its SH groups at the outer surface of the inner membrane. This might correspond to a membrane-Bohr effect, possibly related to the opening of a gating pore in the P_i carrier.     

A meta-analysis revealed insights into the sources,conservation and impact of microRNA 5′-isoforms in four model species

MicroRNA (miRNA) 5′-isoforms, or 5′-isomiRs, are small-RNA species that originate from the same genomic loci as the major miRNAs with their 5′ ends shifted from the 5′ ends of the miRNAs by a few nucleotides. Although 5′-isomiRs have been reported, their origins, properties and potential functions remain to be examined. We systematically studied 5′-isomiRs in human, mouse, fruitfly and worm by analysing a large collection of small non-coding RNA and mRNA profiling data. The results revealed a broad existence of 5′-isomiRs in the four species, many of which were conserved and could arise from genomic loci of canonical and non-canonical miRNAs. The well-conserved 5′-isomiRs have several features, including a preference of the 3p over the 5p arms of hairpins of conserved mammalian miRNAs, altered 5′-isomiRs across species and across tissues, and association with structural variations of miRNA hairpins. Importantly, 5′-isomiRs and their major miRNAs may have different mRNA targets and thus potentially play distinct roles of gene regulation, as shown by an integrative analysis combining miRNA and mRNA profiling data from psoriatic and normal human skin and from murine miRNA knockout assays. Indeed, 18 5′-isomiRs had aberrant expression in psoriatic human skin, suggesting their potential function in psoriasis pathogenesis. The results of the current study deepened our understanding of the diversity and conservation of miRNAs, their plasticity in gene regulation and potential broad function in complex diseases.     

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

Isolation and characterization of major histocompatibility class IIβ genes in an endangered North American cyprinid fish, the Rio Grande silvery minnow (Hybognathus amarus)

The major histocompatibility complex (MHC) is a critical component of the adaptive immune response in vertebrates. Due to the role that MHC plays in immunity, absence of variation within these genes may cause species to be vulnerable to emerging diseases. The freshwater fish family Cyprinidae comprises the most diverse and species-rich group of freshwater fish in the world, but some are imperiled. Despite considerable species richness and the long evolutionary history of the family, there are very few reports of MHC sequences (apart from a few model species), and no sequences are reported from endemic North American cyprinids (subfamily Leuciscinae). Here we isolate and characterize the MH Class II beta genes from complementary DNA and genomic DNA of the non-model, endangered Rio Grande silvery minnow (Hybognathus amarus), a North American cyprinid. Phylogenetic reconstruction revealed two groups of divergent MH alleles that are paralogous to previously described loci found in deeply divergent cyprinid taxa including common carp, zebrafish, African large barb and bream. Both groups of alleles were under the influence of diversifying selection yet not all individuals had alleles belonging to both allelic groups. We concluded that the general organization and pattern of variation of MH class II genes in Rio Grande silvery minnow is similar to that identified in other cyprinid fishes studied to date, despite distant evolutionary relationships and evidence of a severe genetic bottleneck.     

Isolation and characterization of long-chain alkane–degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakır,in the Southeast of Turkey

A strain of long-chain alkane–degrading bacteria, BT1A, was isolated from oil-contaminated soil in Diyarbak?r, in the southeast of Turkey. Morphological, biochemical, and physiological characterization and 16S rRNA gene sequence analysis showed that the strain BT1A was a member of Acinetobacter genus, and it was found to be closely related to Acinetobacter baumannii. The strain BT1A was able to utilize crude petroleum as carbon and energy sources in order to grow. Among the aliphatic hydrocarbons, growth was observed only in the medium containing long-chain alkanes (tridecane, pentadecane, and hexadecane) and squalene. Hexadecane was the most preferred hydrocarbon among the long-chain alkanes. Gas chromatography–mass spectrometry (GC-MS) analysis showed that BT1A degraded 83% of n-alkanes of 1% crude oil in 7 days. The present study indicates that the isolated strain can well be used for biodegradation of hydrocarbons in oil-contaminated sites.     

The crystal and molecular structure of bis(2,2′-dipyridylamine-N,N′)di(nitrato-O)cadmium(II); Solid state 113Cd NMR,components and orientation of the chemical shift tensor relative to the donor ligands

The structure of the titled compound has been determined and refined. The structure consists of isolated molecules separated by ordinary Van der Waals' distances. The Cd atom is on a crystallographic center of symmetry. The coordination polyhedron of the Cd atom is distorted octahedral with four pyridine nitrogen donors in the equatorial plane and with axial oxygen atoms from the nitrate groups. The CdO distance is 2.599(4) Å, the CdN distances are 2.310(4) and 2.316(3) Å, and the NCdN bite angle is 79.0(1)°. The solid state magic angle spinning/cross polarization ¹¹³Cd NMR isotropic chemical shift is +51.4 ppm and the components of the chemical shift tensor are: S₁₁= −92 ppm, S₃₃ = +208 ppm and S₂₂ = +39 ppm and their directions are: in the CdN₄ plane and bisecting the NCdN bite angle, perpendicular to the CdN₄ plane and the third perpendicular to the other two, respectively. This permits the assignment of contributions to the chemical shift tensor of 50 ppm from pyridine nitrogen and of −50 ppm from nitrate oxygen. From the tensor components, atomic nitrogen can be distinguished from aliphatic nitrogen donors. Crystal data: C₂₀H₁₈O₆N₈Cd; M_r = 578.8, F(000) = 580, monoclinic, P2₁/c, a = 8.591(1), b = 16.496(2), c = 7.878(1) Å, β = 95.97°, λ = 0.71073 Å, Mo Kα, V = 1110(1) ų. Z = 2, D_m = 1.73(2), D_x = 1.73 g/cm³, μ = 10.3 cm⁻¹, R_f = 0.039, 1834 reflections, 160 parameters, T ∼ 298 K. Refinement was by full matrix least-squares with anisotropic temperature factors.     

Interactions of daminozide, 2, 2′-dipyridyl,and gibberellins A3, A9 and A20, in stem extension inChrysanthemum morifolium Ramat: an indication that daminozide may not inhibit the responses to GA9 or GA20 by blocking gibberellin hydroxylation

Interactions between the growth retardant daminozide (a substituted succinamic acid) and a subsequent application (1 or 10 g) of either gibberellin A₃, A₉ or A₂₀, on stem extension inChrysanthemum morifolium cv. Bright Golden Anne, indicated that pre-treatment of plants with daminozide largely prevented the response to GA₂₀ as well as to GA₉. The daminozide-GA₃ interaction on total stem length was dependent upon the dose of GA₃ such that, by flowering time, 1 g of GA₃ had virtually eliminated the retardant effect, while 10 g of GA₃ increased stem length to a value similar to that achieved by control shoots receiving 10 g of GA₃. In contrast, prior application of 2, 2-dipyridyl (an inhibitor of hydroxylation in some plant and animal systems) had no significant influence on the time courses of response to any of the GAs. In the absence of daminozide (and 2, 2-dipyridyl) all three GAs were very active in promoting internode extension soon after their application. If 2, 2-dipyridyl can block hydroxylation reactions in chrysanthemum tissues, the results do not support the hypothesis that daminozide restricts GA₉- (or GA₂₀-) induced stem elongation by preventing the hydroxylation of GA₉.     

Synthesis,characterization, and biological assessment of the four stereoisomers of the H3 receptor antagonist 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl)phenyl]benzamide

This Letter describes the asymmetric synthesis of the four stereoisomers (8a–8d) of a potent and highly selective histamine H₃ receptor (H₃R) antagonist, 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl) phenyl]benzamide (1). The physico-chemical properties, in vitro H₃R affinities and ADME of 8a–8d were determined. Stereoisomer 8c (2S,3′S) displayed superior in vitro H₃R affinity over other three stereoisomers and was selected for further profiling in in vivo PK and drug safety. Compound 8c exhibited excellent PK properties with high exposure, desired brain to plasma ratio and reasonable brain half life. However, all stereoisomers showed similar unwanted hERG affinities.