The structure and cytochemistry of the pistil of Hypericum calycinum: the style

Summary A typical style of Hypericum calycinum is solid with a core of transmitting tissue traversing the whole length of the style. This transmitting tissue consists of loosely arranged cells and large intercellular spaces filled with a secretion product. The secretion product is rich in lipids, but poor in proteins and polysaccharides. The intercellular spaces of the transmitting tissue originate partly by a separation of cells as a result of the decomposition of the middle lamella and partly by degeneration of some of the cells of the transmitting tissue. H. calycinum is self-compatible. Both self- and cross-pollinations result in profuse pollen germination on the stigma and pollen tube growth through the style. The data on Hypericum is discussed in relation to information available on other solidstyled systems.     

Unguisin C, a GABA-containing cyclic peptide from the fungus Emericella unguis

Besides the known unguisins A and B, a new cyclic heptapeptide, unguisin C, containing a GABA-derived moiety in the ring, was isolated from the fungus Emericella unguis. The structure was determined by 1D and 2D NMR techniques. Marfey's method was used to determine the absolute stereochemistry. Precursor-directed biosynthesis of the unguisins was performed by supplementation of the culture medium with amino acids ( -Ala, -Ser, -Phe and -Leu). A related cyclic heptapeptide, unguisin D, was detected by HPLC and characterized by sequence analysis using LC-QITMS.     

New triterpenoid saponins from the roots of Gypsophila perfoliata Linn

Nine new triterpenoid saponins (1–9) have been isolated from the roots of Gypsophila perfoliata Linn. Their structures were established on the basis of extensive NMR (¹H, ¹³C, HSQC and HMBC) and ESIMS studies.     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

An efficient strategy for the large-scale synthesis of head-to-tail cyclic peptides

Summary An efficient and generally applicable method for the synthesis of head-to-tail cyclic peptides with HBTU (2-(1H-benzotriazol-1-yl)-1,1,3, 3-tetramethyluronium hexafluorophosphate) has been developed. The utility of this approach was exemplified with the multigram preparation of a potent endothelin receptor-selective (ET_A) antagonist BQ-123 (cyclo[-d-Trp-d-Asp-Pro-d-Val-Leu-]). This methodology can be readily applied to the small-and largescale synthesis of other head-to-tail cyclic peptides.     

Pharmacodynamic synergy strictly dependent on the co-operative aggregation of enantiomers of cyclic peptides in the bacterial cell membrane

The antimicrobial activity of the peptide enantiomers cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] against Bacillus megaterium was investigated. Both these peptides showed very low activity in both an agar diffusion assay and a broth microdilution assay. However, when both peptides were present during the experiments a potent inhibition with an IC(50) value of 2 μM was observed. Furthermore, the peptides also showed low hemolytic activity. Neither peptide had any hemolytic activity in concentrations up to 1mM but when erythrocytes were exposed to both peptides a weak hemolytic activity could be observed with a HC(50) value of 316 μM.     

Computational study of the conformational profiles of model bis-cystine cyclic peptides

Bis-cystine cyclic peptides are a new kind of molecules with potential use as cavitands, transporters or antagonists of target ligands. Studies aimed at establishing their conformational profiles may prove useful in understanding their characteristics and potentiate their use in molecular design. The present investigation reports the results of a computational study devoted to establishing the conformational preferences of model bis-cystine cyclic peptides and the properties in common with their linear analogs. For this purpose a study of four model compounds: (Ac-Cys-X-Cys-NHMe)₂ and (Ac-Cys-X-X-Cys-NHMe)₂ with X = Ala, Val, was performed. The goal of the study was to explore the importance of the conformational nature of the central residues, the effect of the number of them, and the loss of conformational freedom after cyclization on model molecules. Accordingly, the conformational space and the dynamic behaviour of the four cyclic peptides as well as the corresponding linear analogs was carefully explored. The results indicate the existence of structural patterns that might be useful for the use of this kind of molecule in de novo molecular design     

基于分子与形态证据的桃色无心菜(石竹科)分类地位探讨

广义无心菜属(Arenaria L.s.l.)是石竹科(Caryophyllaceae)中分类较为困难的大属之一,基于分子系统学研究结果该属目前已被拆分为多属.基于形态学证据,桃色无心菜(Arenaria melandryoides Edgew.)被置于无心菜属齿瓣亚属[subg.Odontostemma(G.Don)...     

Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-D/L-Val sequence in Gram-positive and Gram-negative bacteria

The melanocortin peptides alpha-MSH, Lys-Pro-Val and Lys-Pro-D-Val are known to be potent anti-inflammatory agents; however their role as antibacterial peptides is less clear. The aim of this study was to determine whether these peptides displayed antibacterial properties, and specifically whether the Lys-Pro-D-Val tripeptide was more potent than Lys-Pro-Val, consistent with their anti-inflammatory actions. alpha-MSH, Ac-Lys-Pro-D-Val-NH2 and Ac-Lys-Pro-Val-NH2 were found to be antibacterial against both Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli) over a broad range of concentrations compared to a control peptide, Ac-Ala-Ala-Ala-NH2. However, the relative potency of alpha-MSH, Ac-Lys-Pro-D-Val-NH2, Ac-Lys-Pro-Val-NH2 did not differ. Furthermore, it was found that the cationic charge on the lysine residue was not required for activity as a variant peptide Ac-Ala-Pro-D-Val-NH2 was also antibacterial. We therefore describe a novel X-Pro-D/L-Val peptide sequence with similarity to the short melanocortin peptides, which possess antibacterial activity. The combined anti-inflammatory and antibacterial action of such peptides may also have potential value therapeutically.     

Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium

Novel inhibitors of fungal ATP-binding cassette transporters were obtained by screening compounds and crude extracts from marine-derived fungi and bacteria using disk diffusion assays of Saccharomyces cerevisiae strains overexpressing a variety of fungal multi-drug efflux pumps. The cyclodepsipeptides unnarmicin A and unnarmicin C were able to sensitize cells overexpressing azole drug pumps ScPdr5p, CaCdr1p, CgCdr1p, and CgPdh1p to sub-MIC concentrations of fluconazole without affecting the growth of CaCdr2p and CaMdr1p overexpressing cells. Unnarmicin A and unnarmicin C were potent inhibitors of rhodamine 6G efflux of CaCdr1p expressing cells with IC₅₀ values of 3.61 and 5.65 μM, respectively. They inhibited the in vitro CaCdr1p ATPase activity at IC₅₀ values of 0.495 and 0.688 μM, respectively. And most importantly, they were able to sensitize azole-resistant Candida albicans clinical isolates to fluconazole. Unnarmicin A and unnarmicin C are candidate efflux pump inhibitors with the potential to be used as adjuvants for antifungal chemotherapy.     

Predicted versus expressed adipokinetic hormones, and other small peptides from the corpus cardiacum-corpus allatum: a case study with beetles and moths

This mass spectrometric study confines itself to peptide masses in the range of 500-1500Da. Adipokinetic hormones (AKHs) that are predicted from the genome of the red flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori, are shown to exist as expressed peptides in the corpora cardiaca (CC) of the respective species as evidenced by various mass spectrometric methods. Additionally, some related species were included in this study, such as the tenebrionid beetles Tribolium brevicornis and Tenebrio molitor, as well as the moths Spodoptera frugiperda, Spodoptera littoralis, Mamestra brassicae and Lacanobia oleracea, to investigate whether AKH peptides are structurally conserved in the same genus or family. Interestingly, the AKH peptide of T. brevicornis is identical to that of T. molitor but not to the ones of its close relative T. castaneum. Moreover, other peptides in T. brevicornis, such as various FXPRL amides (=pyrokinins), also match the complement in T. molitor but differ from those in T. castaneum. All the CC of beetles lacked the signal for the mass of the peptide corazonin. All moths have the nonapeptide Manse-AKH expressed in their CC. In addition, whereas the silk moth has the decapeptide Bommo-AKH as a second peptide, all other moths (all noctuids) express the decapeptide Helze-HrTH. In M. brassicae and L. oleracea a novel amidated Gly-extended Manse-AKH is found as a possible third AKH. The noctuid moth species also all express the same FLRF amide-I, corazonin, and a group-specific isoform of a gamma-PGN-(=gamma-SGNP) peptide. In L. oleracea, however, the latter peptide has a novel sequence which is reported for the first time, and the peptide is code-named Lacol-PK.     

Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

Novel structural class of four disulfide-bridged peptides from Tityus serrulatus venom

A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.     

A novel lysozyme from Xanthomonas oryzae phage varphiXo411 active against Xanthomonas and Stenotrophomonas

In this study, a bacteriophage of Xanthomonas oryzae pv. oryzae designated as varphiXo411 was isolated. Random sequencing of its genome revealed that it is closely related to another X. oryzae phage, Xp10. A cloned fragment carries the lysozyme gene, lys411. The deduced protein, Lys411, shares 92% identity with Xp10 lysozyme, which contains an extra 46 aa at the N-terminus. Lys411 shows over 40% identities to several other phage lysozymes. The His-tagged protein, Lys411H, expressed in Escherichia coli largely formed as inclusion bodies. The insoluble protein was solubilized in urea and purified by passing through a His-bind column, and the lytic activity was then restored by a refolding process. The optimal assay conditions determined for Lys411H are in 0.1M potassium phosphate buffer, pH 6.6 containing 1 mM CuCl(2) at 25 degrees C. Lysis assays using different bacterial cells as the substrates indicate that Lys411H is the first lysozyme active against both Xanthomonas and Stenotrophomonas maltophilia. This suggests that Lys411 can be a candidate to be developed into a therapeutic agent for treating S. maltophilia infections, in addition to the potential use in control of the plant diseases caused by Xanthomonas. By analogy to the situation in Xp10, we predict that varphiXo411 has no holin, the protein required for lysozyme export, and the N-terminal signal-arrest-release sequence of Lys411 can accommodate its own export to the periplasm.     

Anti-Legionella activity of staphylococcal hemolytic peptides

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.     

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-γ than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-γ production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-γ production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.