Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) harms (Bromeliaceae)

A new papain-like cysteine peptidase isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.), has been purified and characterized. The enzyme, named macrodontain I, is the main proteolytic component present in fruit extracts and was purified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narrow saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The alanine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-l-amino acid p-nitrophenyl esters. The N-terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequence similarity to the other pineapple-stem-derived cysteine endopeptidases, being 85.7, 85. 2, and 77.8% identical to comosain, stem bromelain, and ananain, respectively. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.     

Comparison of two cysteine endopeptidases from latices of Morrenia brachystephana Griseb. and Morrenia odorata (Hook et Arn.) Lindley (Asclepiadaceae)

The properties of morrenain b II, a proteinase isolated from the latex of Morrenia brachystephana, were compared with those of morrenain o II, a proteinase obtained from the latex of Morrenia odorata. Both peptidases were purified to homogeneity by acetone precipitation followed by cation exchange chromatography. The enzymes have pI values higher than 9.3 and similar molecular masses (close to 26 kDa) as determined by SDS-PAGE. They display maximum proteolytic activity within an alkaline pH range, and also exhibit esterolytic activity. The N-terminal sequences of morrenain o II and morrenain b II show a high degree of homology between each other and to other cysteine plant proteinases.     

Hieronymain I,a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae)

A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5–9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.     

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N--CBZ-amino acid-p-nitrophenyl esters, and the highest k _cat/K _m values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.     

In vitro regeneration of long spur barrenwort (Epimedium grandiflorum Morr.) from rachis explants

Summary A system for micropropagation of Epimedium grandiflorum Morr. from rachis explants was developed. Explants were cultured onto Murashige and Skoog (MS) basal salts medium supplemented with (per L) 100 mg myo-inositol, 2 mg pyridoxine-HCl, 2 mg nicotinic acid, 0.40 mg thiamine-HCl, 30 g sucrose, and 2 g Phytagel. The medium also contained 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.2, or 0.25 mg/L (0.5, 0.9, or 1.1 μM) combined with either N⁶-benzyladenine (BA) or 2-isopentenyl adenine (2ip) at 2.5, 5, or 10 mg/L (11.1, 22.2, or 44.4 μM BA or 12.3, 24.6, or 49.2 μM 2iP). Cultures were maintained at a 16-h photoperiod (40 μmol/m²/s) and 23±2° C. Callogenesis preceded shoot regeneration. Callus formation increased with higher 2,4-D concentrations. The highest percent regeneration, 83% of explants, was obtained on 10 mg BA per L (44.4 μM) combined with 0.25 mg 2,4-D per L (1.1 μM). The maximum number of shoots, 15 per explant, was obtained from explants cultured on a medium containing 0.1 mg 2,4-D per L (0.45 μM) combined with 2.5 mg BA per L (11.1 μM). Maximum shoot length, 0.4 cm, was obtained on 5 mg BA per L (22.2 μM) combined with 0.2 mg 2,4-D per L (0.9 μM). To produce whole plants, shoots were separated and rooted on hormone-free medium containing 1 g activated charcoal per L. Rachises provided an excellent source of explants for Epimedium micropropagation and proved suitable for callus production.     

Diversity and genetic structure of the Mexican endemic epiphyte Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys (Bromeliaceae)

BACKGROUND AND AIMS: The monoecious, bird-pollinated epiphytic Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys is an endemic bromeliad of the tropical dry forests of Mexico with clonal growth. In the Sierra de Huautla Natural Reserve this species shows a host preference for Bursera copallifera (Sessé & Moc ex. DC) Bullock. As a result of deforestation in the study area, B. copallifera has become a rare tree species in the remaining forest patches. This human-induced disturbance has directly affected the population densities of T. achyrostachys. In this study the genetic consequences of habitat fragmentation were assessed by comparing the genetic diversity, gene flow and genetic differentiation in six populations of T. achyrostachys in the Sierra de Huautla Natural Reserve, Mexico. METHODS: Allozyme electrophoresis of sixteen loci (eleven polymorphic and five monomorphic) were used. The data were analysed with standard statistical approximations for obtaining diversity, genetic structure and gene flow. KEY RESULTS: Genetic diversity and allelic richness were: HE = 0.21 +/- 0.02, A = 1.86 +/- 0.08, respectively. F-statistics revealed a deficiency of heterozygous plants in all populations (Fit = 0.65 +/- 0.02 and Fis = 0.43 +/- 0.06). Significant genetic differentiation between populations was detected (Fst = 0.39 +/- 0.07). Average gene flow between pairs of populations was relatively low and had high variation (Nm = 0.46 +/- 0.21), which denotes a pattern of isolation by distance. The genetic structure of populations of T. achyrostachys suggests that habitat fragmentation has reduced allelic richness and genetic diversity, and increased significant genetic differentiation (by approx. 40 %) between populations. CONCLUSIONS: The F-statistic values (>0) and the level of gene flow found suggest that habitat fragmentation has broken up the former population structure. In this context, it is proposed that the host trees of T. achyrostachys should be considered as a conservation priority, since they represent the limiting factor to bromeliad population growth and connectivity.     

Reproductive biology of Aechmea bracteata (Sw.) Griseb. (Bromelioideae: Bromeliaceae)

• Individuals of Aechmea bracteata show inflorescences with red scape bracts and odourless, yellow, tubular diurnal flowers, with closely arranged sexual organs, producing a large amount of fruits.
• In order to investigate the reproductive system of this species, a suite of characters was assessed: phenology, floral morphology and biology, nectar production dynamics, and fruit and seed production and germination, as a result of controlled pollination crosses. The study was conducted during two flowering seasons in wild populations in Yucatán, Mexico.
• Results suggest an annual flowering pattern with one flowering peak; flowers were diurnal, showing partial dichogamy (protandry)‐herkogamy, anthers and stigma become mature before floral aperture, which could lead to self‐pollination, nectar is produced during anthesis, varying in volume and total sugar concentration during the day; fruits and seeds were produced in all experimental crosses (cross‐pollination, obligated cross‐pollination, assisted and unassisted selfing, geitonogamy and apomixis), as well as high percentage seed germination.
• Several species of Aechmea are reportedly self‐compatible and autogamous, as suggested by results of selfing and non‐assisted selfing crosses, but these results are negated by the presence of apomixis, indicating that the species is apomictic. This is the first report of this breeding system for subgenus Aechmea and the sixth for Bromeliaceae. Polyembryony is here suggested for the first time in this genus and family based on the fact that more seeds were recorded that expected based on ovule numbers. Finally, when performing experimental crosses, estimating reproductive success based on number of seeds is a better approach than number of fruits, due to the effect of pseudogamy.

     

A new species and two new combinations of Ecuadorian Bromeliaceae (Tillandsioideae)

A new species of Guzmania, G. henniae , from southeastern Ecuador is described and illustrated. Two new combinations in the newly erected, segregate genus Racinaea (formerly Tillandsia subgenus Pseudocatopsis), R. dielsii and R. undulifolia are presented and discussed.     

Two new montane vrieseas (Bromeliaceae) from Costa Rica

Two new species ofVriesea with pendent inflorescences,V. uxoris andV. vietoris, are described from the mountains of Costa Rica, and their relationships in the genus are discussed.     

Reproductive biology and natural hybridization between two endemic species of Pitcairnia (Bromeliaceae)

We investigated pollination biology and breeding systems in hybridizing populations of Pitcairnia albiflos and P. staminea; both species are endemic to rocky outcrops at Rio de Janeiro, Brazil. These species are morphologically distinct and easily recognized by floral color: white in P. albiflos and red in P. staminea. Putative hybrids show a large range of intermediate pink floral colors. The showy hermaphroditic flowers offer pollen and nectar that attract many visitors including bees, butterflies, hawk moths, and bats. Although the flowers of both parental species and hybrids open at night, only P. albiflos had other adaptations for nocturnal pollination. Flowering times overlapped during three consecutive years of observation. Bees visited both species and putative hybrids. Cross-pollinations were performed within and among parental species and hybrids in a greenhouse using plants transplanted from the field. Pitcairnia staminea and hybrids are self-compatible and could be spontaneously self-pollinated, whereas P. albiflos, though self-compatible, needs pollinators' services for self-pollination. Facultative agamospermy was found in the parental species. Prezygotic and postzygotic reproductive barriers between these taxa were weak. Reciprocal hand-pollinations between parental species and with hybrids yielded high fruit sets with viable seeds. Evaluations of fruit set, seed set, seed germination, and pollen viability were undertaken to compare the fitness of the hybrids relative to their parents. The hybrids showed equivalent fitness, except for lower pollen viability. Some conservation implications are noted.     

Two new species of Alcantarea (Bromeliaceae,Tillandsioideae) from Brazil

Alcantarea turgida andA. tortuosa are described and illustrated as new species from southeastern Brazil. The relationships ofA. turgida withA. extensa and withA. vinicolor and ofA. tortuosa withA. farneyi and withA. benzingii are discussed.     

Miscellaneous new taxa of Bromeliaceae (XVI)

Four new taxa of Bromeliaceae are described:Billbergia acreana from Brazil;Pitcairnia chocoensis from Colombia; andGuzmania diazii andPitcairnia filifera from Peru. Also included is the new nameWerauhia moralesii from Costa Rica. Miscellaneous new taxa of Bromeliaceae (XV) appeared in Selbyana 21: 125–131. 2000.     

Comparative characterization of two serine endopeptidases from Nocardiopsis sp. NCIM 5124

A protease-producing, crude oil degrading marine isolate was identified as Nocardiopsis sp. on the basis of the morphology, cell wall composition, mycolic acid analysis and DNA base composition. The Nocardiopsis produces two extracellular proteases, both of which are alkaline serine endopeptidases. Protease I was purified to homogeneity by chromatography on CM-Sephadex at pH 5.0 and pH 9.0. Protease II was purified using DEAE-cellulose, Sephadex G-50, phenyl-Sepharose and hydroxyapatite chromatography. Protease I and II had almost similar M(r) of 21 kDa (Protease I) and 23 kDa (Protease II), pI of 8.3 and 7.0 respectively with pH and temperature optima for activity between 10.0 and 11.0 and about 60 degrees C. Specific activities were 152 and 14 U/mg respectively on casein. However, Protease I was antigenically unrelated to Protease II. Both proteases were endopeptidases and required extended substrate binding for catalysis. Both proteases had collagenolytic and fibrinolytic activity but only Protease I had elastinolytic activity. The proteases were chymotrypsin-like with respect to their amino acid compositions and N-terminal sequences.     

我国淡水水华蓝藻-束丝藻属新记录种

由于束丝藻属(Aphanizomenon Mort.ex Born.et Flah.)的藻丝、营养细胞、藻丝末端细胞(Terminal cells)、异型胞(Heterocysts)、厚壁休眠孢子(Akinetes)的形态和大小等特征易变,对鉴定工作造成许多闲难.所以该属的分类一直以来是藻类学者面临的长期难题.基于当今束丝藻属的分类研究,对我国淡水水体束丝藻属进行了研究,比较了该属的藻丝、营养细胞、异形胞、厚壁孢子及末端细胞的特征,发现了我困淡水水体的2个新记录种:柔细束丝藻(Aphanizomenon gracile Lemmermann)和依沙束丝藻(A.issatschenkoi(Usacev)Progkina-Lavrenko),并对其形态特征进行了详细描述.     

Systematics of Tillandsia subgenus Pseudalcantarea (Bromeliaceae)

Tillandsia subg.Pseudalcantarea (Bromeliaceae) includes four species as currently delimited. The phylogenetic relationships of the species in this group are investigated by a cladistic analysis using 16 morphological characters, withVriesea subg.Alcantarea employed as the outgroup.Tillandsia subg.Pseudalcantarea is found to be paraphyletic.Tillandsia grandis andT. paniculata are transferred toTillandsia subg.Tillandsia because they are hypothesized to share a more recent common ancestor withT. utriculata, the type of the genus, than they do with the other two species placed in subg.Pseudalcantarea, i.e.,T. baliophylla andT. viridiflora. These latter two species possibly constitute a basal clade withinTillandsia and are provisionally retained within subg.Pseudalcantarea.     

Two new species of Hechtia (Bromeliaceae,Pitcairnioideae) from western Mexico

Hechtia glauca andHechtia iltisii are described as new and illustrated. Among the Bromeliaceae H. glauca is unusual in its glabrous, glaucous leaf surfaces. Inflorescence and floral dimorphisms are discussed forH. glauca andH. iltisii. A key to species ofHechtia from Novo-Galiciana and adjacent parts of Michoacan is included.     

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.     

Eleutheroside K isolated from Acanthopanax henryi (Oliv.) Harms suppresses methicillin resistance of Staphylococcus aureus

Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin-resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50 µg ml⁻¹. At sub-inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time-kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X-100) or ATPase inhibitor (N, N′-dicyclohexylcarbodiimide). According to western blot analysis, the down-regulated expression of Penicillin-binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose-dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti-MRSA natural drug.     

Revision of the genus Fascicularia Mez (Bromeliaceae)

The genus Fascicularia Mez is revised as part of a study of the Bromeliaceae for the Flora de Chile. Morphological and anatomical investigation of herbarium and living material from cultivation as well as DNA-studies (RAPDs) in cultivated material has led us to conclude that Fascicularia bicolor (Ruiz & Pav.) Mez, the only one species in the genus, comprises two subspecies which are distinguished by their leaf anatomy and morphology.