Delineation of a new structural motif involving NHN γ-turn

Macromolecules are characterized by distinctive arrangement of hydrogen bonds. Different patterns of hydrogen bonds give rise to distinct and stable structural motifs. An analysis of 4114 non-redundant protein chains reveals the existence of a three-residue, (i − 1) to (i + 1), structural motif, having two hydrogen-bonded five-membered pseudo rings (the first, an N H···OC involving the first residue, and the second being N H∙∙∙N involving the last two residues), separated by a peptide bond. There could be an additional hydrogen bond between the side-chain at (i-1) and the main-chain NH of (i + 1). The average backbone torsion angles of −76(±21)° and – 12(±17)° at i creates a tight turn in the polypeptide chain, akin to a γ-turn. Indeed, a search of three-residue fragments with restriction on the terminal C^α···C^α distance and the existence of the two pseudo rings on either side revealed the presence 14 846 cases of a variant, termed NHN γ-turn, distinct from the NHO γ-turn (2032 cases) that has traditionally been characterized by the presence of NHO hydrogen bond linking the terminal main-chain atoms. As in the latter, the newly identified γ-turns are also of two types—classical and inverse, occurring in the ratio of 1:6. The propensities of residues to occur in these turns and their secondary structural features have been enumerated. An understanding of these turns would be useful for structure prediction and loop modeling, and may serve as models to represent some of the unfolded state or disordered region in proteins.     

Effect of single pyrrole replacement with β-alanine on DNA binding affinity and sequence specificity of hairpin pyrrole/imidazole polyamides targeting 5′-GCGC-3′

N-Methylpyrrole (Py)–N-methylimidazole (Im) polyamides are small organic molecules that can recognize predetermined DNA sequences with high sequence specificity. As many eukaryotic promoter regions contain highly GC-rich sequences, it is valuable to synthesize and characterize Py–Im polyamides that recognize GC-rich motifs. In this study, we synthesized four hairpin Py–Im polyamides 1–4, which recognize 5′-GCGC-3′ and investigated their binding behavior with surface plasmon resonance assay. Py–Im polyamides 2–4 contain two, one, and one β-alanine units, replacing the Py units of 1, respectively. The binding affinities of 2–4 to the target DNA increased 430, 390, and 610-fold, respectively, over that of 1. The association and dissociation rates of 2 to the target DNA were improved by 11 and 37-fold, respectively, compared with those of 1. Interestingly, the association and dissociation rates of 3 and 4 were higher than those of 2, even though the binding affinities of 2, 3, and 4 to the target DNA were comparable to each other. The binding affinity of 2 to DNA with a 2 bp mismatch was reduced by 29-fold, compared with that to the matched DNA. Moreover, the binding affinities of 3 and 4 to the same mismatched DNA were reduced by 270 and 110-fold, respectively, indicating that 3 and 4 have greater specificities than 2 and are suitable as DNA-binding modules for engineered epigenetic regulation.     

Design of environmentally sensitive fluorescent 2′-deoxyguanosine containing arylethynyl moieties: Distinction of thymine base by base-discriminating fluorescent (BDF) probe

We have synthesized various substituted 8-arylethynylated 2′-deoxyguanosine derivatives. Among them, acetyl substituted deoxyguanosine analogue 4c showed a remarkable solvent dependent fluorescence property, that is, an intense fluorescence in non-polar solvents but extremely weak fluorescence in polar solvents like methanol. By using solvatofluorochromic deoxyguanosine analogue 4c, we have developed highly thymine (T) selective fluorescent DNA probes that can sense T opposite 4c in a target DNA regardless of the flanking sequences. We were able to demonstrate that 4c can be used as a T specific base-discriminating fluorescent (BDF) nucleoside in homogeneous fluorescence assay.     

Design and synthesis of a novel anthracene-based fluorescent probe through the application of the Suzuki–Miyaura cross-coupling reaction

We report on a simple synthetic route to a novel anthracene-based bis-armed amino acid derivative as a useful fluorescent probe. Various photophysical studies of this amino acid derivative are also described. Here, Suzuki-Miyaura cross-coupling reaction has been used as a key step for carbon-carbon bond formation.     

Regiospecificity and stereospecificity in the enzymatic conjugation of glutathione with (±)-benzo(a)pyrene 4,5-oxide

¹³C-NMR analysis of the glutathione conjugates formed from (±)-benzo(a)-pyrene 4,5-oxide-4,5-¹³C by a purified glytathione transferase from little skate ($\text{Raja erinacea}$) liver demonstrated that equivalent amounts of the positional isomers (4,5-dihydro-4-hydroxy-5-glutathionylbenzo(a)pyrene and 4,5-dihydro-4-glutathionyl-5-hydroxybenzo(a)pyrene) were formed. Separation of these conjugates by HPLC and subsequent ¹³C-NME studies showed that only one diastereoisomer of each positional isomer was formed by the skate enzyme, each enantiomer of the arene oxide having produced only one of the two possible positional isomers. The non-enzymic reaction of (±)-benzo(a)pyrene 4,5-oxide with glutathione produced the four possible stereoisomers resulting from $\text{trans}$ addition to the epoxide ring. This was also true when rat liver cytosol was used as the source of transferase activity. The data demonstrate that skate liver glutathione transferase 4 has high substrate regiospecificity and stereospecificity for (±)-benzo(a)pyrene 4,5-oxide.     

Design of commissioning process for Halcyon™ linac with a new rigid board: A clinical experience

PurposeThis study performed the accurate measurements of beam profiles with a new rigid board, which was consistent with the supplied reference beam profiles (RBPs) for clinical Halcyon model.MethodsPercentage depth doses (PDDs), lateral and diagonal dose profiles were measured and compared with RBPs. A water tank was set on the rigid board bridged Halcyon bore without sagging and source-to-surface distance was 90.0 cm. Field sizes were from 2.0 to 28.0 cm squares and depths of lateral and diagonal dose profiles were 1.3, 5.0, 10.0, and 20.0 cm. For the PDD, the depth of maximum dose (d_max), PDD value at depth of 10.0 cm (PDD₁₀), and absolute dose difference (DD) between RBP and measured beam profiles (MBP) were evaluated. For lateral and diagonal dose profiles, DDs for the whole and divided areas (central, shoulder, and extended areas) defined by third derivative, and distance-to-agreement (DTA) in the penumbra area were evaluated.ResultsFor PDDs, the differences of d_max and PDD₁₀ and DD beyond the d_max were within 1.0 mm, 0.3%, and 1.0%, respectively. For lateral and diagonal dose profiles, the DDs reached approximately 5.0% in the whole area because of penumbra area, while the DDs in the central, shoulder, and extended areas were within 1.0%, 2.0%, and 1.0%, respectively. The DTAs in the penumbra area were within 0.8 mm.ConclusionsThe supplied RBPs can be used clinically owing to the good agreement with the accurate MBPs with rigid board.     

4′-Alkoxy derivatives of abscisic acid: a promising new probe for mechanisms of abscisic acid perceptions

The preparation of new compounds for exploring the molecular andbiochemical mechanisms of abscisic acid signal perception is described. The4-carbonyl group of abscisic acid was replaced with either benzyloxy orn-octyloxy groups. Both modified products possessedABA-like activity in bioassays using barley aleurone protoplasts. The benzyloxyderivative was active in dehydrin induction tests, but not in inhibition of-amylase induction tests. The difference in the activity of this chemical inthe two assay systems suggests that the perception system for the inhibition ofamylase induction systems differs from that of dehydrin induction.     

Radwaste at sea: A new era of polarization or a new basis for consensus?

Abstract

A coalition of third world nations, led by the Pacific island countries and those European nations who have developed land‐based disposal programs for their radioactive wastes, seek to amend the London Convention on Dumping (the international treaty controlling ocean disposal of radioactive and other wastes) in order to ban ocean disposal of low‐level radioactive wastes. Pro‐dumping nations maintain that the treaty may only be amended based on science and that current scientific research indicates that low‐level waste represents neither a threat to the integrity of the marine environment nor human health. Anti‐dumping nations, on the other hand, argue that the same science, particularly the models used to predict the fate and the effects of these wastes, exhibits sufficient uncertainty to preclude judgments about the absence of harm from future disposal activities. These differing conclusions mirror differing assessments of risk. These assessments build on the differing social, political, and economic values placed on use of the ocean and on conflicting conceptions of the fundamental rights and obligations of nations whose use of the ocean may impinge on the resources of others. Each side's continued intransigence may result in unilateral ocean disposal activities with serious consequences for the London Convention on Dumping (LDC) and its control over other wastes transported to sea for disposal. Initiatives of anti‐dumping nations to expand the LDC's decision‐making framework to examine the social, economic, and political issues underlying each side's interpretation of scientific evidence offer hope to address the underlying non‐scientific issues and perhaps to strengthen decision‐making within the LDC.     

2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy

Abstract

Reactive oxygen species (ROS) are critically important chemical intermediates in biological studies, due to their multiple physiologically essential functions and their often pathologically deleterious effects. Consequently, it is vital that their presence in biological samples has to be quantifiable. However, their high activity, very short life span and extremely low concentrations make ROS measurement a scientifically challenging subject for researchers. One of the widespread methods for ROS detection, based on the oxidation of the non-fluorescent probe 2′,7′-dichlorodihydrofluorescein (DCFH₂) to yield the highly fluorescent 2′,7′-dichlorofluorescein (DCF), was developed more than 40 years ago. However, from its initial application, argumentative questions have arisen regarding its action mechanisms, reaction principles and especially its specificity. Herein, the authors attempt to undertake a comprehensive review: to describe the basic characteristics of DCFH₂; to discuss the present views of the mechanisms of its fluorescence formation; to summarize the fluorescence formation interferents; to outline its application in biological research; and to underline its advantages and disadvantages in ROS detection as well as for the methodological considerations that arise during analysis.     

‘Anticalins’: a new class of engineered ligand-binding proteins with antibody-like properties

The development of soluble receptor proteins that recognise given target molecules — ranging from small chemical compounds to macromolecular structures at a cell surface, for example — is of ever increasing importance in the life sciences and biotechnology. For the past century this area of application was dominated by antibodies, which were traditionally generated via immunisation of animals but have recently also become available by means of protein engineering methods. The so-called ‘anticalins’ offer an alternative type of ligand-binding proteins, which has been constructed on the basis of lipocalins as a scaffold. The central element of this protein architecture is a β-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities. The bilin-binding protein (BBP) was employed as a model system for the preparation of a random library with 16 selectively mutagenized residues. Using bacterial phagemid display and colony screening techniques, several lipocalin variants — termed anticalins — have been selected from this library, exhibiting binding activity for compounds like fluorescein or digoxigenin. Anticalins possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Compared with them, they exhibit however several advantages, including a smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Apart from haptenic compounds as targets, anticalins should also be able to recognise macromolecular antigens, provided that the random library is accordingly designed. Hence, they should not only serve as valuable reagents for bioanalytical purposes, but may also have a potential in replacing antibodies for medical therapy.     

Systematics of the Pacific monkey‐faced bats (Chiroptera: Pteropodidae), with a new species of Pteralopex and a new Fijian genus

The fruit‐bat genus Pteralopex comprises the monkey‐faced bats, a group of six endangered species found only in old‐growth forests on certain islands in the south‐west Pacific (the Solomon Islands and Fiji). The taxonomy of the genus is reviewed in detail and updated accordingly. Two ‘cryptic’ biological species are shown to occur in sympatry on both Bougainville and Choiseul in the northern Solomon Islands (corresponding to Pteralopex anceps Andersen, 1909 and a previously undescribed species) and each is accordingly described and reviewed. A new genus (Mirimiri) is erected for the Fijian monkey‐faced bat (formerly Pteralopex acrodonta), which differs greatly both morphologically and genetically from species of Pteralopex in the Solomon Islands. Ecomorphological differences between sympatric Pteralopex species are briefly reviewed, including potential differences in functional morphology and feeding ecology. Geographic patterns of occurrence and future survey priorities for monkey‐faced bats are also discussed.     

Refined crystal structure of γ-chymotrypsin at 1.9 Å resolution: Comparison with other pancreatic serine proteases

The crystal structure of γ-chymotrypsin, the monomeric form of chymotrypsin, has been determined and refined to a crystallographic R-factor of 0.18 at 1.9 Å resolution. The details of the catalytic triad involving Asp102, His57 and Ser195 agree well with the results found for trypsin (Chambers & Stroud, 1979) and Streptomyces griseus protease A (Sielecki et al., 1979). As in many of the other serine proteases, the Oγ of Ser195 does not appear to be hydrogen-bonded to His57.The three-dimensional structures of γ- and α-chymotrypsin (Birktoft & Blow, 1972) are closely similar. The largest backbone differences occur in the “calcium binding loop” (residues 75 to 78) and in the “autolysis loop” (residues 146, 149 and 150). Ala149 and Asn150 are disordered in γ-chymotrypsin, whereas they are stabilized by intermolecular interactions in α-chymotrypsin. The conformation of Ser218 is also different, presumably the indirect result of the dimeric interactions of α-chymotrypsin. These results are discussed in terms of the slow, pH-dependent interconversion of α- and γ-chymotrypsin.     

Synthesis of a quinazoline derivative: a new α₁-adrenoceptor ligand for conjugation to quantum dots to study α₁-adrenoceptors in living cells

Quantum dots (QDs) that are conjugated to small molecule derivatives of drugs and endogenous ligands may be useful tools to study the distribution and dynamic of membrane bound receptors, ion channels and transporters in live cells. In order to use these tools, it is necessary to functionalize QDs with bioactive ligands. In this paper, we successfully synthesized a ligand of α(1)-adrenoceptor that could be conjugated to QDs. In addition, the conjugation of the ligands to QDs and their biological activity were evaluated through binding assay with 30 nM QD conjugates in living human embryonic kidney 293 cells.     

Design and synthesis of a new sialyl Lewis X mimetic: how selective are the selectin receptors?

The present paper reports the molecular modeling-based design and synthesis of an optically pure noncarbohydrate mimetic of sialyl Lewis X to inhibit E-selectin. Biological evaluation of the designed substance as well as that of its enantiomer gave, contrary to expectations, comparable IC50 values. Results are discussed in terms of receptor binding specificity and the molecular modeling protocol used.     

What's in a name? Aschersonia insperata: a new pleoanamorphic fungus with characteristics of Aschersonia and Hirsutella

A new anamorphic species from a Philippine tropical forest occurs as reddish-orange to orange, tuberculate stromata on unidentified homopteran larvae, and produces both Aschersonia and Hirsutella-like synanamorphs. A molecular phylogenetic analysis was conducted to determine the most appropriate generic placement for this fungus. Based on its phylogenetic relationships, a comparison of the complexity and persistence of each anamorph, and the speculated relevance of each synanamorph to survival, we describe the new fungus as Aschersonia insperata sp. nov.     

Green fluorescent protein – Tagged HCV non-enveloped capsid like particles: Development of a new tool for tracking HCV core uptake

Circulating ‘free’ non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core₁₇₃-eGFP, eGFP-core₁₉₁ and eGFP-core₁₇₃ can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells – the main target of HCV – but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.     

Is the abundance of species determined by their functional traits? A new method with a test using plant communities

The relation between functional traits and abundance of species has the potential to provide evidence on the mechanisms that structure local ecological communities. The niche-limitation/limiting-similarity hypothesis, derived from MacArthur and Levins’ original concept, predicts that species that are similar to others in terms of functional traits will suffer greater competition and hence be less abundant. On the other hand, the environment-filtering/habitat-optimum hypothesis predicts that groups of species with functional traits that are close to the optimum for that environment, and are therefore similar to other species, will be more abundant. We propose a new niche-assembly model for predicting the relative abundance of species in communities from their functional traits, which can detect the patterns that would be expected from either of these hypotheses. The model was fitted to eight plant communities sampled in the Lake Ohau district of New Zealand. For seven of the sites, the patterns could not be distinguished from that expected under a null model. However, in one site there was highly significant departure from the null model in the direction expected from the niche-limitation hypothesis. The site was probably the most productive of those examined. It is possible that competition for light rather than belowground resources, or faster recovery from disturbance, allowed greater predictability. Surprisingly, the predictability was seen when just the presences of a species’ neighbours in trait space were taken into account, but not when the potential effects of those neighbours were weighted by their abundance. For three of the four model types, the effects of species on each other were consistently negative: a significant trend. These results contradict the various neutral models of ecological communities.     

Vietnamese species of Taumacera viridis–group with a description of new species (Coleoptera: Chrysomelidae: Galerucinae)

The species of Taumacera viridis species–group from Vietnam are revised. Four species are recognized. Taumacera indica (Jacoby, 1889), T. occipitalis (Laboissière, 1933) and T. variceps (Laboissière, 1933), are redescribed. Taumacera phuquoca sp. nov. from Phu Quoc National Park (southern Vietnam) is described. An identification key is provided for all Vietnamese species.www.zoobank.org/urn:lsid:zoobank.org:pub:396862F9-8A47-4101-A27C-CD0625E68FA1.     

Apples increase nitric oxide production by human saliva at the acidic pH of the stomach: a new biological function for polyphenols with a catechol group?

Dietary inorganic nitrate is secreted in saliva and reduced to nitrite by bacterial flora. At the acidic pH of the stomach nitrite is present as nitrous acid in equilibrium with nitric oxide (*NO), and other nitrogen oxides with nitrating and nitrosating activity. *NO in the stomach exerts several beneficial effects, but nitrosating/nitrating species have been implicated as a possible cause of epithelial neoplasia at the gastroesophageal junction. We investigated the effects of apple extracts on *NO release by human saliva at pH 2. A water extract obtained from apple homogenate increased *NO release caused by acidification of saliva. Data show that polyphenols were responsible for this activity, with chlorogenic acid and (+)-catechin the most active and concentrated species. However, ferulic acid, a hydroxycinnamic acid with only one aromatic hydroxyl group, did not increase *NO release. Fructose, the most representative sugar in apples, was also inactive. Interestingly, ascorbic acid in saliva induced a SCN(-)-enhanced burst of *NO but, unlike apple, the release was transient. The simultaneous addition of ascorbic acid and apple extract caused a burst of *NO followed by the increased steady-state level characteristic of saliva containing apple extract. Chlorogenic acid and (+)-catechin, but not ferulic acid, formed o-semiquinone radicals and nitrated polyphenols, suggesting the scavenging of *NO(2) by o-semiquinones. Our results propose that some apple polyphenols not only inhibit nitrosation/nitration but also promote *NO bio-availabilty at the gastric level, a previously unappreciated function.