Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Amplitude-time characteristics of the long-latency components (<Emphasis Type="Italic">N</Emphasis>1, <Emphasis Type="Italic">N</Emphasis>2, and <Emphasis Type="Italic">P</Emphasis>300) of acoustic evoked potential in healthy subjects

The aim of the study was to trace the cumulative changes in the amplitude-time parameters of the AEP (acoustic evoked potential) components N1, N2, and P300 in the experimental situations of different complexities (counting and listening to sounds) and to compare the differences between young and mature subjects. The AEP was recorded in 12 healthy subjects aged 18 to 22 years and in 12 subjects aged 32 to 59 years. It was revealed that the components N1, N2, and P300 recorded in the situation of listening without any preliminary instruction did not differ in young and mature individuals. In the younger subjects, a higher N1 amplitude was revealed when they counted sounds, compared to listening, whereas the latency values of this component did not differ, irrespective of the complexity of the task. The component N2 was shown to demonstrate latency stability in relation to the age and the experimental situation. The N2 amplitude was higher in the situation of counting sounds in both age groups. The amplitude-time parameters of P300 did not differ in subjects of different ages in the listening task. The features of the components N1, N2, and P300 revealed in young and mature subjects in the experimental situations differing in complexity lead us to suggest that, with age, humans develop adaptive mechanisms contributing to successful task performance.     

<Emphasis Type="Italic">N</Emphasis>-glycan moieties of the crustacean egg yolk protein and their glycosylation sites

Vitellogenin (Vg) is the precursor of the egg yolk glycoprotein of crustaceans. In the prawn Macrobrachium rosenbergii, Vg is synthesized in the hepatopancreas, secreted to the hemolymph, and taken up by means of receptor-mediated endocytosis into the oocytes. The importance of glycosylation of Vg lies in its putative role in the folding, processing and transport of this protein to the egg yolk and in the fact that the N-glycan moieties could provide a source of carbohydrate during embryogenesis. The present study describes, for the first time, the structure of the glycan moieties and their sites of attachment to the Vg of M. rosenbergii. Bioinformatics analysis revealed seven putative N-glycosylation sites in M. rosenbergii Vg; two of these glycosylation sites are conserved throughout the Vgs of decapod crustaceans from the Pleocyemata suborder (N ¹⁵⁹ and N ⁶⁶⁰). The glycosylation of six putative sites of M. rosenbergii Vg (N ¹⁵¹, N ¹⁵⁹, N _,¹⁶⁸ N _,⁶¹⁴ N ⁶⁶⁰ and N ²³⁰⁰) was confirmed; three of the confirmed glycosylation sites are localized around the N-terminally conserved N-glycosylation site N ¹⁵⁹. From a theoretical three-dimensional structure, these three N-glycosylated sites N ¹⁵¹, N ¹⁵⁹, and N ¹⁶⁸ were localized on the surface of the Vg consensus sequence. In addition, an uncommon high mannose N-linked oligosaccharide structure with a glucose cap (Glc₁Man₉GlcNAc₂) was characterized in the secreted Vg. These findings thus make a significant contribution to the structural elucidating of the crustacean Vg glycan moieties, which may shed light on their role in protein folding and transport and in recognition between Vg and its target organ, the oocyte.     

Effective number of breeding adults in Oregon spotted frogs (<Emphasis Type="Italic">Rana</Emphasis><Emphasis Type="Italic">pretiosa</Emphasis>): genetic estimates at two life stages

We used genetic methods to estimate the effective number of breeders (N _b) in a population of Rana pretiosa, an imperiled amphibian in western North America. Microsatellite data was gathered from large samples of adults, eggs, and juveniles collected in 2006. We wished to determine where in the life cycle the greatest reductions in N _b occur, and to compare genetic estimates of N _b to an egg mass count estimate of the number of breeding adults. We predicted that N _b estimated at the metamorph stage would be reduced by increased variance in family size due to egg mass mortality. Contrary to our prediction, estimates of N _b at the egg and metamorph stages were similar. Thus, we found no evidence of inflated variance in family size between the two stages. If our results for this population are typical for R. pretiosa, then increased variance in family size during the egg to metamorph stage may not be a strong factor in reducing the effective population sizes (N _e) relative to the census sizes (N) in this species.     

Photosynthetic and transpiration responses of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. plants to <Emphasis Type="Italic">ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.     

Modulation of Carbohydrate Metabolism During <Emphasis Type="Italic">N</Emphasis>-Methyl <Emphasis Type="Italic">N</Emphasis>-Nitrosourea Induced Neurotoxicity in Mice: Role of Curcumin

The present study was carried out to elucidate the effectiveness of curcumin in mitigating the adverse effects caused by N-Methyl N-Nitrosourea (MNU) on mouse cerebellum and cerebrum. Male laca mice received either intravenous MNU treatment at a dose of 10 mg/kg bw in sterile double distilled water, curcumin alone 60 mg/kg bw in drinking water, or combined MNU and curcumin treatment on alternate days for a period of 2 months. The effects of different treatments were studied on carbohydrate metabolizing enzymes viz: hexokinase, glucose-6-phosphatase (G6P), glucose-6-isomerase (G6I), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and glycogen levels. Curcumin supplementation to MNU treated mice was able to reduce significantly the activities of the G6P, G6I, hexokinase, LDH, SDH and increased the glycogen contents in both the regions of brain which were altered following MNU treatment. Hence, curcumin shall prove to be effective in ameliorating the adverse effects caused by MNU.     

<Emphasis Type="Italic">N</Emphasis>-Linked glycoengineering for human therapeutic proteins in bacteria

Approx. 70% of human therapeutic proteins are N-linked glycoproteins, and therefore host cells for production must contain the relevant protein modification machinery. The discovery and characterisation of the N-linked glycosylation pathway in the pathogenic bacterium Campylobacter jejuni, and subsequently its functional transfer to Escherichia coli, presents the opportunity of using prokaryotes as cell factories for therapeutic protein production. Not only could bacteria reduce costs and increase yields, but the improved feasibility to genetically control microorganisms means new and improved pharmacokinetics of therapeutics is an exciting possibility. This is a relatively new concept, and progress in bacterial N-glycosylation characterisation is reviewed and metabolic engineering targets revealed.     

The diagnostic implications of the separation of<Emphasis Type="Italic">Entamoeba histolytica</Emphasis> and<Emphasis Type="Italic">Entamoeba dispar</Emphasis>

For much of the last hundred years most cases of amoebiasis have been diagnosed by light microscopy. Only relatively recently have we become aware that this technique is usually incapable of distinguishing between two species-Entamoeba histolytica andE. dispar-only the first of which is a pathogen. The implications of this for patient management and, even more, for the validity of epidemiological surveys, are only slowly being addressed. What is clear is that methods are urgently required to distinguish between infections with these two species and this review attempts to summarise some of those, which have been developed to meet this need.     

Mapping <Emphasis Type="Italic">O</Emphasis>-GlcNAc modification sites on tau and generation of a site-specific <Emphasis Type="Italic">O</Emphasis>-GlcNAc tau antibody

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-d-glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.     

Recombination within mouse <Emphasis Type="Italic">t</Emphasis> haplotypes has replaced significant segments of <Emphasis Type="Italic">t</Emphasis>-specific DNA

Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1–2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the “complete” t haplotypes t ^w2 , t ⁰ , t ^w32 , t ^w71 , and t ^w75 . We show that only t ^w32 is a true “complete” t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within t ^w71 and t ^w75 mice.     

An on-column derivatization method for the determination of homocysteine-thiolactone and protein <Emphasis Type="Italic">N</Emphasis>-linked homocysteine

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with ε-amino group of a protein lysine residue generating N-Hcy-protein. This reaction impairs and alters protein’s function and has been implicated in atherothrombotic disease. Here, we describe new high-performance liquid chromatography assays for the determination of Hcy-thiolactone, protein N-linked Hcy, and Hcy based on an on-column derivatization with o-phthaldialdehyde and fluorescence detection. The on-column derivatization generates narrow peaks, which allows fast run times (3–5 min) and facilitates determination of N-linked Hcy directly from acid hydrolysates of plasma protein. Utility of these assays was demonstrated with human urine and plasma samples.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Flow-cytometry assay for apoptosis using fluorophor 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange

Recently it has been shown that redox catalytic interactions of cytochrome c with cardiolipin (CL) and subsequent oxidation of CL occurs during apoptosis. Oxidation of CL is accompanied by a release of cytochrome c from the mitochondria into the cytoplasm, a key event in development of apoptosis. 10-N-Nonyl acridine orange (NAO), a fluorophor that forms a stable complex with reduced form of CL, but not with oxidized CL, can be used for flow cytometry analysis of this effect. It has been shown that after the incubation of CTLL-2 cells in the presence of 7% ethanol (90 min) and subsequent staining with NAO, a cell population with low intensity of fluorescence appears. Flow cytometry analysis of the cells by means of a conventional method (annexin V-FITC/propidium iodide (PI)) showed that the cell population with low intensity of NAO fluorescence corresponded to the population of apoptotic cells with good coincidence of percentages. Then apoptosis was induced in the cells of three lines by growth factor deprivation (IL-2-dependent cell line CTLL-2) or as a result of actinomycin D treatment, an RNA synthesis inhibitor (Jurkat and Raji cell lines). Comparison of the data obtained by a conventional assay (annexin V-FITC/PI) and a newly elaborated assay (NAO/PI) has demonstrated a good coincidence. The data obtained with these methods exhibited significant level of correlation (0.953, p < 0.0001).     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

Hormonal control of the outgrowth of axillary buds in <Emphasis Type="Italic">Alstroemeria</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

We study apical dominance in Alstroemeria, a plant with an architecture very different from the model species used in research on apical dominance. The standard explant was a rhizome with a tip and two vertically growing shoots from which the larger part had been excised leaving ca. 1 cm stem. The axillary buds that resumed growth were located at this 1-cm stem just above the rhizome. They were released by removal of the rhizome tip and the shoot tips. Replacement of excised tips by lanolin with indole-3-butyric acid (IBA) restored apical dominance. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-napthylphthalamic acid (NPA) reduced apical dominance. 6-Benzylaminopurine (BAP) enhanced axillary bud outgrowth but the highest concentrations (> 9 μM) caused fasciation. Thidiazuron (TDZ) did not show improvement relative to BAP. Even though the architecture of Alstroemeria and the model species are very different, their hormonal mechanisms in apical dominance are for the greater part very similar.     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.