Inhibition of HIV-1 and M-MLV reverse transcriptases by a major polyphenol (3,4,5 tri-O-galloylquinic acid) present in the leaves of the South African resurrection plant, Myrothamnus flabellifolia.

A polyphenol-rich extract of the medicinal resurrection plant Myrothamnus flabellifolia was shown to inhibit viral (M-MLV and HIV-1) reverse transcriptases. Fractionation and purification of this extract yielded the major polyphenol, 3,4,5 tri-O-galloylquinic acid, as the main active compound. A sensitive, ethidium bromide based fluorescent assay, was developed and used to monitor the kinetics of M-MLV and HIV-1 reverse transcriptases in the presence and absence of 3,4,5 tri-O-galloylquinic acid. Kinetic monitoring of these enzymes in the presence of 3,4,5 tri-O-galloylquinic acid revealed non-competitive inhibition with IC(50) values of 5 µM and 34 µM for the M-MLV and HIV-1 enzymes, respectively. We propose that 3,4,5 tri-O-galloylquinic acid and related polymers have potential as indigenous drugs for anti-viral therapy.

Towards a systems-based understanding of plant desiccation tolerance.

Vegetative desiccation tolerance occurs in a unique group of species termed 'resurrection plants'. Here, we review the molecular genetic, physiological, biochemical, ultrastructural and biophysical studies that have been performed on a variety of resurrection plants to discover the mechanisms responsible for their tolerance. Desiccation tolerance in resurrection plants involves a combination of molecular genetic mechanisms, metabolic and antioxidant systems as well as macromolecular and structural stabilizing processes. We propose that a systems-biology approach coupled with multivariate data analysis is best suited to unraveling the mechanisms responsible for plant desiccation tolerance, as well as their integration with one another. This is of particular relevance to molecular biological engineering strategies for improving plant drought tolerance in important crop species, such as maize (Zea mays) and grapevine (Vitis vinifera).

An overview of the biology of the desiccation-tolerant resurrection plant Myrothamnus flabellifolia.

BACKGROUND: Myrothamnus flabellifolia is unique as the only woody resurrection plant. It is an important plant in southern Africa because of its widespread occurrence and usage in African medicine and traditional culture. Many reports have investigated facets of its biology and the mechanisms associated with its desiccation tolerance. SCOPE: The general biology of the woody resurrection plant Myrothamnus flabellifolia is reviewed. The review focuses on the geography and ecology, systematic placement, evolution, morphology and reproductive ecology of M. flabellifolia as well as the wood anatomy and re-filling mechanism. In addition, the desiccation tolerance, ethnobotanical importance and medicinal properties of the plant are reviewed. Also, future research avenues are suggested, in particular the necessity to research the biogeography and systematics of the species and the role of the polyphenols present, as well as the molecular basis of the plant's desiccation tolerance.

ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation.

ASP53, a 53 kDa heat soluble protein, was identified as the most abundant protein in the mature seeds of Acacia erioloba E.Mey. Immunocytochemistry showed that ASP53 was present in the vacuoles and cell walls of the axes and cotyledons of mature seeds and disappeared coincident with loss of desiccation tolerance. The sequence of the ASP53 transcript was determined and found to be homologous to the double cupin domain-containing vicilin class of seed storage proteins. Mature seeds survived heating to 60°C and this may be facilitated by the presence of ASP53. Circular dichroism spectroscopy demonstrated that the protein displayed defined secondary structure, which was maintained even at high temperature. ASP53 was found to inhibit all three stages of protein thermal denaturation. ASP53 decreased the rate of loss of alcohol dehydrogenase activity at 55°C, decreased the rate of temperature-dependent loss of secondary structure of haemoglobin and completely inhibited the temperature-dependent aggregation of egg white protein.

Response of the leaf cell wall to desiccation in the resurrection plant Myrothamnus flabellifolius.

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.

The South African and Namibian populations of the resurrection plant Myrothamnus flabellifolius are genetically distinct and display variation in their galloylquinic acid composition.

The polyphenol contents and compositions in desiccated leaves of Myrothamnus flabellifolius plants collected in various locations in Namibia and South Africa were analyzed using UV spectroscopy and high-performance liquid chromatography-mass spectrometry. A study of the genetic relatedness of these populations was also performed by determination of the DNA sequence of the intergenic spacer region between the psbA and the trnH genes in the chloroplast genome. Namibian M. flabellifolius plants contained significantly more polyphenols than South African plants. Namibian plants essentially contained a single polyphenol, 3,4,5-tri-O-galloylquinic acid, whereas South African plants contained a variety of galloylquinic acids including 3,4,5-tri-O-galloylquinic acid together with higher molecular weight galloylquinic acids. Sequence analysis revealed a 1.4% divergence between Namibian and South African plants corresponding to the separation of these populations of approximately 4 x 10(6) years. The significance of the poly-phenol content and composition to the desiccation tolerance of the two populations is discussed.

Development of plant regeneration and transformation protocols for the desiccation-sensitive weeping lovegrass Eragrostis curvula.

A tissue culture protocol, suitable for transformation, was established for the pasture grass Eragrostis curvula. Callus was generated in the dark from leaf and seed tissues on a medium comprising MS salts supplemented with 2 mg/l 2,4-D, 0.01 mg/l BAP and 2% sucrose. Plant regeneration occurred via organogenesis on the same medium with 6% and 3% sucrose for shoot and root formation, respectively. Optimal regeneration (50 plantlets per callus) occurred when light of 45 micromol/m2 per s was used. The yeast Saccharomyces cerevisiae Hsp12 gene was cloned into the Sac1 of the pCAMBIAUbeeQ vector and callus was transformed by biolistic bombardment. Best transformation (30%) occurred when the target tissue was bombarded twice at a distance of 70 mm using a bombardment pressure of 9,100 kPa. Although successful transformation and transcription of the Hsp12 gene occurred, no Hsp12 protein was found present in tissue extracts of transformed grass.

The predominant polyphenol in the leaves of the resurrection plant Myrothamnus flabellifolius, 3,4,5 tri-O-galloylquinic acid, protects membranes against desiccation and free radical-induced oxidation.

The predominant (>90%) low-molecular-mass polyphenol was isolated from the leaves of the resurrection plant Myrothamnus flabellifolius and identified to be 3,4,5 tri-O-galloylquinic acid using 1H and 13C one- and two-dimensional NMR spectroscopy. The structure was confirmed by mass spectrometric analysis. This compound was present at high concentrations, 44% (by weight) in hydrated leaves and 74% (by weight) in dehydrated leaves. Electron microscopy of leaf material fixed with glutaraldehyde and caffeine demonstrated that the polyphenols were localized in large vacuoles in both hydrated and dehydrated leaves. 3,4,5 Tri-O-galloylquinic acid was shown to stabilize an artificial membrane system, liposomes, against desiccation if the polyphenol concentration was between 1 and 2 microg/mug phospholipid. The phase transition of these liposomes observed at 46 degrees C was markedly diminished by the presence of 3,4,5 tri-O-galloylquinic acid, suggesting that the presence of the polyphenol maintained the membranes in the liquid crystalline phase at physiological temperatures. 3,4,5 Tri-O-galloylquinic acid was also shown to protect linoleic acid against free radical-induced oxidation.

LEA (late embryonic abundant)-like protein Hsp 12 (heat-shock protein 12) is present in the cell wall and enhances the barotolerance of the yeast Saccharomyces cerevisiae.

Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions. Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity. The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall. This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage.

Significant quantities of the glycolytic enzyme phosphoglycerate mutase are present in the cell wall of yeast Saccharomyces cerevisiae.

NaOH was used to extract proteins from the cell walls of the yeast Saccharomyces cerevisiae. This treatment was shown not to disrupt yeast cells, as NaOH-extracted cells displayed a normal morphology upon electron microscopy. Moreover, extracted and untreated cells had qualitatively similar protein contents upon disruption. When yeast was grown in the presence of 1 M mannitol, two proteins were found to be present at an elevated concentration in the cell wall. These were found to be the late-embryogenic-abundant-like protein heat-shock protein 12 and the glycolytic enzyme phosphoglycerate mutase. The presence of phosphoglycerate mutase in the cell wall was confirmed by immunocytochemical analysis. Not only was the phosphoglycerate mutase in the yeast cell wall found to be active, but whole yeast cells were also able to convert 3-phosphoglycerate in the medium into ethanol, provided that the necessary cofactors were present.

Determination of species-specific components in the venom of Parabuthus scorpions from southern Africa using matrix-assisted laser desorption time-of-flight mass spectrometry.

The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Parabuthus (Pocock 1890) by means of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) and to construct a species-specific venom code for species identification. The venom compositions of sixteen Parabuthus species, occurring in southern Africa, were characterized using representative peaks in the molecular mass range of 6400-8400 Da. This mass range is characteristic for the typical long-chain neurotoxins influencing sodium channels. Only a few of these peptides have been sequenced up to now. The impetus for development of these species-specific profiles was the observation of unique, highly reproducible mass spectral peaks within a specific species. An identification label for all the different species could be found using a minimum number of peaks. MALDI-TOFMS is therefore proposed as a complementary method to morphological and behavioural characteristics for species and ultimately subspecies discrimination.