Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells. The levels of dihydrofolate reductase mRNA as well as the enzyme activity also increase when both cell lines are serum stimulated at the permissive temperature. When ts13 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity declines rapidly and dihydrofolate reductase mRNA is below detectable levels. On the contrary, when tsAF8 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity increases and mRNA levels are detectable slightly above G0 levels, even though the cells are blocked in the G1 phase. Studies with 2 other cDNA clones (one with an insert whose expression is cell cycle dependent and the other with an insert whose expression is not cell cycle dependent) indicate that the results are not due to aspecific toxicity or the effect of temperature. We conclude that the expression of different genes is affected differently by the ts block in G1, even when these genes are all growth-related.     

Approaches to breeding for salinity tolerance - a case study on Porteresia coarctata

Cereals are the world's major source of food for human nutrition. Among these, rice (Oryza sativa) is the most prominent and represents the staple diet for more than two-fifths (2.4 billion) of the world's population, making it the most important food crop of the developing world (Anon., 2000a). Rice production in vast stretches of coastal areas is hampered due to high soil salinity. This is because rice is a glycophyte and it does not grow well under saline conditions. In order to increase rice production in these areas there is a need to develop rice varieties suited to saline environments. Research has shown that Porteresia coarctata, a highly salt tolerant wild relative of rice growing in estuarine soils, is an important material for transferring salt tolerant characteristics to rice. It is quite possible that Porteresia may be used as a parent for evolving better and truly salt resistant varieties. The inadequate results and the difficulties associated with conventional breeding techniques necessitate the use of the tools of crop biotechnology in unravelling some of the characteristics of Porteresia that have been highlighted in this report. In view of the limited resources available for increasing salinity tolerance to the breeders to wild rice germplasm, Porteresia is undoubtedly one of the key source species for elevating salinity tolerance in cultivated rice.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Promoter-independent regulation of vimentin expression in mammary epithelial cells by val(12)ras and TGFbeta

The 1,029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val(12)ras. These cell lines respond to TGFbeta by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFbeta. In vitro, vimentin expression is induced in all the cell lines by TGFbeta treatment, whereas cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFbeta is mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFbeta treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half-life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFbeta, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.     

Induced root-secreted phenolic compounds as a belowground plant defense

Rhizosphere is the complex place of numerous interactions between plant roots, microbes and soil fauna. Whereas plant interactions with aboveground organisms are largely described, unravelling plant belowground interactions remains challenging. Plant root chemical communication can lead to positive interactions with nodulating bacteria, mycorriza or biocontrol agents or to negative interactions with pathogens or root herbivores. A recent study¹ suggested that root exudates contribute to plant pathogen resistance via secretion of antimicrobial compounds. These findings point to the importance of plant root exudates as belowground signalling molecules, particularly in defense responses. In our report,² we showed that under Fusarium attack the barley root system launched secretion of phenolic compounds with antimicrobial activity. The secretion of de novo biosynthesized t-cinnamic acid induced within 2 days illustrates the dynamic of plant defense mechanisms at the root level. We discuss the costs and benefits of induced defense responses in the rhizosphere. We suggest that plant defense through root exudation may be cultivar dependent and higher in wild or less domesticated varieties.Key words: root exudates, plant defense, t-cinnamic acid, fusarium, induced defensePlants grow and live in very complex and changing ecosystems. Because plants lack the mobility to escape from attack by pathogens or herbivores, they have developed constitutive and in addition inducible defenses that are triggered by spatiotemporally dynamic signaling mechanisms. These defenses counteract the aggressor directly via toxins or defense plant structures or indirectly by recruitment of antagonists of aggressors. Whereas induced defenses are well described in aboveground interactions, evidence of the occurrence of such mechanisms in belowground interactions remains limited. The biosynthesis of a defensive molecule could be both constitutive and inducible with a low level of a preformed pool (Fig. 1). In addition, upon encounter of an attacking organism, those levels could be induced to rise locally to a high level of active compound that is able to disarm the pathogen.^2,3 Only a few examples show that root exudates play a role in induced plant defense. Hairy roots of Ocimum basilicum secrete rosmarinic acid only when challenged by the pathogenic fungus Pythium ultimum.⁴ Wurst et al.⁵ reported on the induction of irridoid glycosides in root exudates of Plantago lanceolata in presence of nematodes. In vivo labelling experiments² with ¹³CO₂ showed the induction of phenolic compounds secreted by barley roots after Fusarium graminearum infection and the de novo biosynthesis of root secreted t-cinnamic acid within 2 days. These results show that the pool of induced t-cinnamic acid originated from both pre-formed and newly formed carbon pools (Fig. 1), highlighting a case of belowground induced defense inside and outside the root system.Open in a separate windowFigure 1Suggested mechanisms for the induction of root defense exudates in barley in response to Fusarium attack. Upon pathogen attack by Fusarium, the initial preformed pool of phenolic compounds is increased by the addition of inducible, de novo biosynthesized t-cinnamic acid. Both, the preformed pool and the de novo biosynthesized pool fuel the exudation of defense compounds from infected roots.The concept of fitness costs is frequently presented to explain the coexistence of both constitutive and induced defense.⁶ In the case of induced defense, resources are invested in defenses only when the plant is under attack. In the absence of an infection, plants can optimize allocation of their resources to reproduction and growth to compete with neighbours.⁷ Constitutive defenses are thought to be more beneficial when the probability of attack is high, whereas adjustable, induced defenses are more valuable to fight against an unpredictable pathogen. Non disturbed soil is a heterogeneous matrix where biodiversity is very high and patchy^8,9 and organism motility is rather restricted.¹⁰ As a consequence of the patchiness, belowground environment is expected to be favourable to selection for induced responses.¹¹ The absence of defense root exudates between two infections may form an unpredictable environment for soil pathogens and reduce the chance for adaptation of root attackers. Plants may also use escape strategies to reduce the effect of belowground pathogens. Henkes et al. (unpublished) showed that Fusarium-infected barley plants reduced carbon allocation towards infected roots within a day and increased allocation carbon to uninfected roots. These results illustrate how reallocation of carbon toward non infected root parts represents a way to limit the negative impact of root infection.We have demonstrated the potential of barley plants to defend themselves against soil pathogen by root exudation.² Even the barley cultivar ‘Barke’ used in our study, a modern cultivated variety, was able to launch defense machinery via exudation of antimicrobial compounds when infected by F. graminearum. We suggest that plant defense through root exudation might be cultivar dependent and perhaps higher in wild or less domesticated varieties. Taddei et al.¹² reported that constitutivelyproduced root exudates from a resistant Gladiolus cultivar inhibit spore germination of Fusarium oxysporum whereas root exudates from a susceptible cultivar do not affect F. oxysporum germination. Root exudates from the resistant cultivar contained higher amounts of aromaticphenolic compounds compared to the susceptible cultivar and these compounds may be responsible for the inhibition of spore germination. Metabolic profiling of wheat cultivars, ‘Roblin’ and ‘Sumai3’, respectively, susceptible and resistant to Fusarium Head Blight, showed that t-cinnamic acid was a discriminating factor responsible for resistance/defense function.¹³ Therefore it is likely that wild barley varieties hold higher defense capacities compare to cultivated varieties selected for high yield. In the future, plant breeders in organic and low-input farming could use root-system defense ability as new trait in varietal variation.     

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan^® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-10⁶ copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.