Evidence for folate-salvage reactions in plants

Folates in vivo undergo oxidative cleavage, giving pterin and p-aminobenzoylglutamate (pABAGlu) moieties. These breakdown products are excreted in animals, but their fate is unclear in microorganisms and unknown in plants. As indirect evidence from this and previous studies strongly suggests that plants can have high folate-breakdown rates (approximately 10% per day), salvage of the cleavage products seems likely. Four sets of observations support this possibility. First, cleavage products do not normally accumulate: pools of pABAGlu (including its polyglutamyl forms) are equivalent to, at most, 4-14% of typical total folate pools in Arabidopsis thaliana, Lycopersicon esculentum and Pisum sativum tissues. Pools of the pterin oxidation end-product pterin-6-carboxylate are, likewise, fairly small (3-37%) relative to total folate pools. Second, little pABAGlu built up in A. thaliana plantlets when net folate breakdown was induced by blocking folate synthesis with sulfanilamide. Third, A. thaliana and L. esculentum tissues readily converted supplied breakdown products to folate synthesis precursors: pABAGlu was hydrolysed to p-aminobenzoate and glutamate, and dihydropterin-6-aldehyde was reduced to 6-hydroxymethyldihydropterin. Fourth, both these reactions were detected in vitro; the reduction used NADPH as cofactor. An alternative salvage route for pABAGlu, direct reincorporation into dihydrofolate via the action of dihydropteroate synthase, appears implausible from the properties of this enzyme. We conclude that plants are excellent organisms in which to explore the biochemistry of folate salvage

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

BACKGROUND: Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. METHODS: We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. RESULTS: In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. CONCLUSION: We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma

Proteomic Modeling for HIV-1 Infected Microglia-Astrocyte Crosstalk

Background: HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous system’s microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease. Methods and Findings: MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species. Conclusions: These observations provide unique insights into glial crosstalk during disease by supporting astrocytemediated regulation of microglial function and its influence on the onset and progression of neuroAIDS. The results open new insights into previously undisclosed pathogenic mechanisms and open the potential for biomarker discovery an

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements

Detailed structural investigation of the grafting of [Ta(=CHtBu)(CH(2)tBu)(3)] and [Cp*TaMe4] on silica partially dehydroxylated at 700 degrees C and the activity of the grafted complexes toward alkane metathesis

The reaction of [Ta(=CHtBu)(CH(2)tBu)(3)] or [Cp*Ta(CH3)(4)] with a silica partially dehydroxylated at 700 degreesC gives the corresponding monosiloxy surface complexes [(=SIO)Ta(=CHtBu)(CH(2)tBu)(2)] and [(=SiO)Ta(CH3)(3)Cp*] by eliminating a a-bonded ligand as the corresponding alkane (H-CH(2)tBu or H-CH3). EXAFS data show that an adjacent siloxane bridge of the surface plays the role of an extra surface ligand, which most likely stabilizes these complexes as in [(equivalent toSiO)Ta(=CHtBu)(CH(2)tBU)(2)(equivalent toSiOSiequivalent to)] (1a') and [(equivalent toSiO)Ta(CH3)(3)Cp*(equivalent toSiOSiequivalent to)] (2a'). In the case of [(=SiO)Ta(equivalent toCHtBu)(CH(2)tBu)(2)(equivalent toSiOSiequivalent to)], the structure is further stabilized by an additional interaction: a C-H agostic bond as evidenced by the small J coupling constant for the carbenic C-H (J(C-H) = 80 Hz), which was measured by J-resolved 2D solid-state NMR spectroscopy. The product selectivity in propane metathesis in the presence of [(=SIO)Ta(equivalent toCHtBu)(CH(2)tBu)(2)(equivalent toSiOSiequivalent to)] (1a') as a catalyst precursor and the inactivity of the surface complex [(equivalent toSIO)Ta(CH3)(3)Cp*(equivalent toSiOSiequivalent to)] (2a') show that the active site is required to be highly electrophilic and probably involves a metallacyclobutane intermediate