Effective/census population size ratio estimation: a compendium and appraisal

With an ecological-evolutionary perspective increasingly applied toward the conservation and management of endangered or exploited species, the genetic estimation of effective population size (N_e) has proliferated. Based on a comprehensive analysis of empirical literature from the past two decades, we asked: (i) how often do studies link N_e to the adult census population size (N)? (ii) To what extent is N_e correctly linked to N? (iii) How readily is uncertainty accounted for in both N_e and N when quantifying N_e/N ratios? and (iv) how frequently and to what degree might errors in the estimation of N_e or N affect inferences of N_e/N ratios? We found that only 20% of available N_e estimates (508 of 2617; 233 studies) explicitly attempted to link N_e and N; of these, only 31% (160 of 508) correctly linked N_e and N. Moreover, only 7% (41 of 508) of N_e/N ratios (correctly linked or not) reported confidence intervals for both N_e and N; for those cases where confidence intervals were reported for N_e only, 31% of N_e/N ratios overlapped with 1, of which more than half also reached below N_e/N = 0.01. Uncertainty in N_e/N ratios thus sometimes spanned at least two orders of magnitude. We conclude that the estimation of N_e/N ratios in natural populations could be significantly improved, discuss several options for doing so, and briefly outline some future research directions.     

Blockade by N-methylhydroxylamine or activation of guanylate cyclase and elevations of guanosine 3′,5′-monophosphate levels in nervous tissues

Hydroxylamine and N-methylhydroxylamine prevented the activation of soluble guanylate cyclase by the endogenous activator as well as by nitroso compounds such as N-methyl-N′-nitro-N-nitroguanidine or nitroprusside, while the other derivaties of hydroxylamine were ineffective. Hydroxylamine and N-methylhydroxylamine did not alter the basal guanylate cyclase activity of purified enzyme preparations. Kinetics analysis indicated that N-methylhydroxylamine competes with N-methyl-N′nitro-N-nitrosuguanidine for guanylate cyclase. The activation of guanylate cyclase by N-methyl-N′-nitro-N-nitrosoguanidine and its inhibition by N-methylhydroxylamine were reversible reactions. These efects of N-methyl-N′-nitro-N-nitrosoguanine and N-methylhydroxylamine were observed with guanylate cyclase from other tissues.N-Methylhydroxylamine preveneed the increase of guanosine 3′,5′-monophosphate (cyclic GMP) levels in cerebellar slices of guinea pig by N-methyl-N′-nitro-N-nitroguanidine, veratridine and adenosine, while the elevalations of adenosine 3′,5′-monophosphate by these agents were not affected. N-Methylhyroxylamine also blocked the increased of cyclic GMP levels by carbachol, prostaglandin E₁ and N-methyl-N′-nitro-N-nitrosoguanidine in neuroblastoma N1E 115 cells. Thus N-methylhydroxylamine prevents the activation of guanylate cyclase and the increased synthesis of cyclic GMP in responses to transmitters without blocking the synthesis of cyclic GMP via basal enzyme activity.     

N-acetyl conjugates of basic amino acids newly identified in rat urine

Ninhydrin-negative conjugates of basic amino acids were isolated from rat urine and were characterized. The following conjugates of basic amino acids are the compounds newly identified in animal urine specimens, N^α-acetyl-N^π-methylhistidine, N^α-(N-acetyl-β-alanyl)histidine (N-acetylcarnosine), N^α-acetyl-N^G,N^′G-dimethylarginine, N^α-acetyl-N^G,N^G-dimethylarginine, and N^α-acetyl-N^?,N^?,N^?-trimethyllysine.     

Conversion of N-acetylglucosamine to CMP-N-acetylneuraminic acid in a cell-free system of rat liver

A soluble fraction of rat liver converts glucosamine and N-acetylglucosamine in the presence of ATP and UTP to N-acetylneuraminic acid. This system, when supplemented with CTP, forms CMP-N-acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP-N-acetylglucosamine and N-acetylneuraminic acid. Kinetic analysis reveals N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, N-acetylmannosamine, N-acetylmannosamine 6-phosphate and N-acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N-acetylglucosamine to N-acetylmannosamine was seen.     

Versatile coordination behavior of N,N-di(alkyl/aryl)-N′-benzoylthiourea ligands: Synthesis, crystal structure and cytotoxicity of palladium(II) complexes

The synthesis and characterization of Pd(II) complexes with the general formula cis-[Pd(L-O,S)₂] (HL = N,N-diethyl-N′-benzoylthiourea, N,N-diisobutyl-N′-benzoylthiourea or N,N-dibenzyl-N′-benzoylthiourea) and trans-[PdCl₂(HL-S)₂] (HL = N,N-diphenyl-N′-benzoylthiourea, N,N-di-n-butyl-N′-benzoylthiourea or N,N-diisopropyl-N′-benzoylthiourea) are reported. These complexes were formed from the reaction between PdCl₂ and N,N-di(alky/aryl)-N′-benzoylthiourea in acetonitrile with the formulation dependent on the nature of HL. The new Pd(II) complexes have been characterized by analytical and spectral (FT-IR, UV-Vis, ¹H NMR and ¹³C NMR, Mass) techniques. The molecular structures of two of the complexes (1 and 5) have been conformed by X-ray crystallography. Complex 1 shows cytotoxicity against human breast cancer cells.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

The influence of dietary calcium and phosphorus on intestinal calcium transport in rats given vitamin D metabolites.

A new method for the simple analysis of methylated amino acids based on autoradiography is introduced. With this technique a survey of protein methylation in a prokaryote, Escherichia coli, and a eukaryote, fibroblasts in culture, was carried out in an attempt to identify, quantitate, and determine the subcellular localization of all the methylated amino acids found in the proteins of these organisms.In mammalian cells using an established mouse fibroblast line (3T3), we have found that nuclei-free and mitochondria-free cytoplasm contain readily detectable amounts of four identifiable methylated amino acids: N^?,N^?-dimethyllysine, N^?,N^?,N^?-trimethyllysine, N^G,N^G-dimethylarginine (or N^G-methylarginine), and N^G,N′^G-dimethylarginine. The crude nuclear pellet also contains these methylated amino acids, but in addition contains N^?-methyllysine and a new as yet unidentified methylated compound. Histones purified from these nuclei contain essentially the same array of methylated compounds.The ribosomal subunits of the mammalian cells contained only small amounts of the methylated amino acids; the 40S subunit contained a substantial amount of just one, N^G,N^G-dimethylarginine (or N^G-methylarginine), and smaller amounts of N^G,N′^G-dimethylarginine, and an as yet unidentified methylated compound. The 60S subunit contained even smaller amounts of methylated amino acids, 50% of which was N^?,N^?,N^?-trimethyllysine and smaller amounts of N^?-methyllysine, N^?,N^?-dimethyllysine, and N^G,N^G-dimethylarginine. These subunits also contained an as yet unidentified methylated compoundThese results were in marked contrast to those that we obtained with the prokaryote, Escherichia coli. Only the proteins of the 50S ribosomal subunit of the bacteria contained methylated amino acids. Of those present 50% was N^?,N^?,N^?-trimethyllysine, with the remainder distributed about equally between N^?-methyllysine and three unknowns, one of which is apparently the same as that found in the 60S subunit of the mouse fibroblasts. All of the N^?-methyllysine was apparently in the small acidic proteins, L7 and L12.     

Cytokinin activity of N-phenyl-N′-(4-pyridyl)urea derivatives

We have synthesized 35 N-phenyl-N′-(4-pyridyl)urea derivatives and tested their cytokinin activity in the tobacco callus bioassay. Among them, N-phenyl-N′- (2-chloro-4-pyridyl)urea is highly active, the optimum concentration of which is lower than 4 × 10^?9 M (0.001 ppm), 3 compounds, i.e. N-(2-methylphenyl)-N′-(2-chloro-4-pyridyl)urea, N-(3-methylphenyl)-N′-(2-chloro-4-pyridyl)urea and N-(3-chlorophenyl)-N′-(2-chloro-4-pyridyl) urea are as active as N⁶-benzyladenine (concentration for optimum yield: 4.4 × 10^?8 M or 0.01 ppm), and N-phenyl-N′-(2-methyl-4-pyridyl)urea and N-(2-chlorophenyl)-N′-(2-chloro-4-pyridyl)urea are as active as N-phenyl-N′-(4-pyridyl)urea (concentration for optimum yield: 4.7 × 10^?7 M or 0.1 ppm), while the activity of the other 29 compounds are not so remarkable and 11 of them are almost or completely inactive.     

Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2

Sirtuins catalyze the NAD⁺ dependent deacetylation of N^ε-acetyl lysine residues to nicotinamide, O′-acetyl-ADP-ribose (OAADPR) and N^ε-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N^ε-modified lysine containing inhibitors against SIRT1 and SIRT2. N^ε-Selenoacetyl and N^ε-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N^ε-thioacetyl group. The N^ε-3,3-dimethylacryl and N^ε-isovaleryl moieties gave significant inhibition in comparison to the N^ε-acetyl group present in the substrates. In addition, the studied N^ε-alkanoyl, N^ε-α,β-unsaturated carbonyl and N^ε-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N^ε-modification. These results are applicable for further screening of N^ε-acetyl analogues.     

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.     

The effects of N-substitution of chitosan and the physical form of the products on the rate of hydrolysis by chitinase from Streptomyces griseus

N-Formyl, N-chloroacetyl, N-glycyl, N-isobutyryl, and N-pentanoyl derivatives of chitosan have been prepared. N-Acetylchitosan was the derivative most susceptible to chitinase from Streptomyces griseus and lysozyme from chicken egg-white, but the susceptibility was not restrictive. The relative rates of hydrolysis by chitinase with respect to R in the RCONH group were CH₃ > CH₃CH₂ > H > CH₃CH₂CH₂ > (CH₃)₂CH > NH₂CH₂ > ClCH₂. Neither enzyme hydrolysed chitosan or its N-methylene, N-benzylidene, N-benzoyl, N-nicotinyl, and N-fatty acyl (C₅C₁₈) derivatives, and lysozyme did not hydrolyse N-butyrylchitosan. N-Acetylhexanoyl-chitosans, which had d.s. ratios of ~0.7: ~0.3 and ~0.3; ~0.7, were hydrolysed at ~0.75 and ~0.04 of the rate of N-acetylchitosan (powder) by chitinase. O-Acylation of N-acylchitosans caused a decrease in the rates of hydrolysis by chitinase. N-Acetylchitosan gels were hydrolysed at 8–13 times the rate for crab-shell chitin. These results indicate that not only N- and O-substituents but also the physical form of the substrates influence the rates of hydrolysis by these enzymes.     

Induction of mitotic gene conversion in Saccharomyces cerevisiae by N-nitrosated pesticides

The herbicide N-(2-benzothiazolyl)-N′-methylurea (Benzthiazuron, Gatnon®) the insecticides 2-isopropoxyphenyl-N-methylcarbamate (Propoxur, Unden®) and 1-naphthyl-N-methylcarbamate (Carbaryl, Sevin®), together with their N-nitroso derivatives, were examined for genetic activity. A diploid strain of Saccharomyces cerevisiae heteroallelic at the gene loci ade2 and trp5, was used to test for the induction of mitotic gene conversion in these two unlinked gene loci. The non-nitrosated compounds had no influence on the frequency of mitotic gene conversions. The nitrosated substances, however, displayed marked convertogenic activities. N-nitrosopropoxur (N-nitrosopropoxur, N-nitrosocarbaryl) were much more active than the substituted N nitrosomethylurea (N-nitrosobenzthiazuron). N-Nitrosocarbaryl induced the greatest number of mitotic gene conversions.     

A kinetic analysis of peroxidase-catalyzed N,N-dimethylaniline dimerization

The horseradish peroxidase/hydrogen peroxide-supported oxidation of N,N-dimethylaniline is shown to proceed along three separate primary pathways; N-demethylation to N-metnylaniline, dimerization to N,N,N′,N′-tetramethyl-p,p′-benzidine and its oxidation products, and another dimerization to an unidentified water-soluble product, possibly an N-oxide. The first pathway predominates at low concentrations of peroxidase, while the dimerizations predominate at higher enzyme concentrations. These studies were made possible by the development of a very sensitive fluorimetric assay for N,N,N′,N′-tetramethyl-p,p′-benzidine. Kinetic studies of the dimerizations show them to be second-order with peroxidase concentration at low enzyme concentrations (less than 0.1 μg/3 ml), but near first order at higher enzyme concentrations. The dimerizations appear to involve interactions between pairs of enzyme-substrate or enzyme-intermediate complexes.     

12-Substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridines as novel topoisomerase I-targeting antitumor agents

2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine and a few of its 12-substituted analogs are active as TOP1-targeting agents. Studies were performed to further evaluate the potential of this series of non-camptothecin TOP1-targeting agents. The influence of a hydroxymethyl, formyl, N,N-dimethylaminomethyl, 2-(N,N-dimethylamino)ethyl, 3-(N,N-dimethylamino)propyl), and 4-(N,N-dimethylamino)butyl substituent at the 12-position on TOP1-targeting activity and tumor cell growth was evaluated. In addition, the relative pharmacologic activities of the 12-carboxamide analog, as well as its N-methyl and N,N-dimethyl derivatives were assessed.     

Metabolism of acetylpolyamines by monoamine oxidase,diamine oxidase and polyamine oxidase

N¹-Monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N⁸-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N¹,N¹²-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N¹-monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine are attacked by polyamine oxidase.     

Nucleophilic displacement reactions of some N-acetyl-N-aryl-β-d-xylopyranosylamines

The 4-O-methanesulphonyl (and toluene-p-sulphonyl), 3,4-di-O-methanesulphonyl (and toluene-p-sulphonyl), and 3,4-di-O-benzoyl-2-O-methanesulphonyl derivatives of N-acetyl-N-p-methoxyphenyl- and N-acetyl-N-p-chlorophenyl-β-d-xylopyranosylamine have been synthesised together with the N-acetyl-N-p-methoxyphenyl and N-acetyl-N-p-chlorophenyl derivatives of 3,4-di-O-benzoyl-2-O-methanesulphonyl-β-d-lyxopyranosylamine. The relative reactivity of the hydroxyl groups of the N-acetyl-N-aryl-β-d-xylopyranosylamines towards sulphonylation has been established. On heating the 2- and 4-mesylates of N-acetyl-N-aryl-β-d-xylopyranosylamines and the 2-mesylate of N-acetyl-N-aryl-β-d-lyxopyranosylamines with sodium azide in N,N-dimethylformamide or acetonitrile, either nucleophilic replacement of the mesyl groups of their solvolysis with participation of the N-acetyl group occurred. In this way, β-d-xylo compounds were converted into α-l-arabino and β-d-lyxo derivatives.     

Comparison of mutagenicity and chemical properties of N-methyl-N′-alkyl-N-nitrosoureas

Thed mutagenic activities of 11 N-methyl-N′-alkyl-N-nitrosoureas were tested on Samonellatyphimurium TA1535 and compared with chemical properties (alkylating activity and decompostion rate). In their relative mutagenicities the N-nitrosoureas that had a cyclic N′-alkyl group showed far more mutagenic activity than those having a chain N′-alkyl group. M(1-A)NU and M(2-A)NU, which had the most bulky N′-alkyl group in this series, exhibited lethal effects at high concentrations. The mutagenicity showed a small positive correlation with decomposition rates but not with alkylating activities on 4-(p-nitrobenzyl_prridine. The highest mutagenicity in this series was observed in N-methyl-N′-cyclobutyl-N-nitrosourea.These results suggest that, in this series of N-methyl-M′-alkyl-N-nitrosoureas, structural differences in the N′-alkyl groups had great significance in mutagenicity.     

Identification of N-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway in Bifidobacterium longum

We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Mutagenic and inhibitory properties of some new purine analogs on Salmonella typhimurium TA1530

Mutagnic and growth-inhibitory effects of 2-amino-N⁶-hydroxyadenine, 2-amino-N⁶-methoxyadenine and 2-amino-N⁶-methyl-N⁶-hydroxyadenine were examined on S. typhimurium TA1530. All compounds showed strong mutagenic activity, and 2-amino-N⁶-hydroxyadenine induced mutations at a dose as low as 1 μg/ml. 2-Amino-N⁶-hydroxyadenine and, to a lesser extent, 2-amino-N⁶-methyl-N⁶-hydroxyadenine (but not 2-amino-N⁶-methoxyadenine) exerted inhibitory effects on bacterial growth. The specificity of mutagenic action of these base analogs, as well as the target for reversion of the his⁻ marker in TA1530, is discussed.