The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY). In E. coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain) with an α-helical domain on its N terminus (N domain). The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques. Methods to describe the reaction kinetically are presented. The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases. The intrinsic guanine nucleotide dissociation rates of FtsY are about 10⁵ times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor. Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase–nucleotide exchange factor complex not only in its structure but also kinetically. The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor. From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.     

Validation of a GC–FID method for rapid quantification of nicotine in fermented extracts prepared from Nicotiana tabacum fresh leaves and studies of nicotine metabolites

A new GC–FID method, which allows rapid and reliable quantitation of nicotine in tobacco leaf extracts, was developed and validated. To avoid nicotine adsorption on the column, an amine-deactivated capillary column was used. The method developed was applied to study the degradation of nicotine in a fermented aqueous extract, and a loss of nearly 20% of nicotine over 12 months was observed. Careful inspection of GC–MS runs from concentrated samples of the same extract revealed the presence of nicotine metabolites such as nornicotine, anatabine, myosmine, 2,3′-bipyridyl, and 2-pyrrolidinone.     

Flavonol quantification and stability of phenolics in fermented extracts from fresh Betula pendula leaves

An HPLC method, which allows reliable quantitation of flavonols and other phenolics in birch leaf extracts, was developed and validated. The method was applied to study the bioconversion of flavonols in fermented aqueous extracts. Almost 100% of the flavonols were converted during the 12 months observation period. The generated phenolics as well as consecutive conversion products were identified by HPLC–DAD, LC–MS and GC–MS techniques.     

Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica

We examined the molecular mechanisms of the cytotoxicity of Entamoeba histolytica, using the loss of transepithelial electrical resistance (TER) of monolayers of Madin-Darby canine-kidney (MDCK) cells on their incubation with axenic trophozoites of the HM1-IMSS strain. Such loss of TER occurs very early (in 2–5 min) and is caused by the opening of tight junctions and the detachment of cells. We used specific inhibitors for three of the four molecules currently accepted as being responsible for cytotoxicity: galactose-specific adhesin(s), phospholipase A, and cysteine proteinases. We also used inhibitors of calcium channels. Axenic trophozoites of E. histolytica strain HM1-IMSS were preincubated with the different inhibitors for 1 h prior to their coincubation with MDCK-cell monolayers. The only inhibitor that effectively blocked the loss of TER caused by the parasite was galactose. We suggest that in this experimental model, galactose-specific adhesin(s) are essential for amebic cytotoxicity. Received: 8 June 1999 / Accepted: 21 July 1999     

Population Pharmacokinetics of Mycophenolic Acid

Clinical Pharmacokinetics - Objective: The pharmacokinetics of mycophenolic acid (MPA) were compared in renal transplant patients receiving either mycophenolate mofetil (MMF) or enteric-coated...     

Granule cell number, cell death and cell proliferation in the dentate gyrus of wild-living rodents

Adult neurogenesis in the dentate gyrus occurs at species-specific levels. Wood mice (Apodemus flavicollis) show higher proliferation rates than laboratory mice and voles (Clethrionomys glareolus, Microtus subterraneus). We compare rates of cell death and proliferation and investigate if cell proliferation leads to the long-term recruitment of granule cells. Granule and pyknotic cell numbers were estimated in wild-living rodents in different age classes and compared with laboratory mice of mixed genetic background. All species differ significantly in their number of granule cells, except for the comparison of laboratory mice with European pine voles. Granule cell number is significantly higher in old bank voles and wood mice as compared to adults (23 and 37%, respectively). The number of pyknotic cells is highest in wood mice and lowest in laboratory mice. Across all species, the numbers of proliferating and pyknotic cells correlate. Despite differences in cell proliferation and cell death, the ratio of proliferating to pyknotic cells does not differ between adults of the wild-living species, but in laboratory mice a significantly lower proportion of cells die compared with the other species. In addition, the ratio of proliferating to pyknotic cells was significantly higher in old wood mice than in adults. We conclude (i) that cell proliferation can lead to an increase in granule cell number in wild-living rodents and (ii) that species- and age-specific changes of the ratio between proliferating and pyknotic cells occur as deviations from a close correlation of these two numbers across all species and age groups.     

SCG/Kinjoh mice: a model of ANCA-associated crescentic glomerulonephritis with immune deposits

BACKGROUND: Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice spontaneously develop crescentic glomerulonephritis (CGN), systemic vasculitis, and perinuclear ANCA (pANCA), and have been suggested as an animal model for human antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AASV). Since no systematic serologic, immunohistologic, or structural evaluation had been performed thus far, we reinvestigated the development of ANCA and CGN in these mice. METHODS: SCG/Kj mice were subjected to serologic and urinary analysis, as well as histologic evaluation of the kidneys by standard light, immunofluorescence, and electron microscopy at regular intervals during the course of the disease. RESULTS: Perinuclear ANCA developed as early as the 6th week of life, increasing both in frequency and titer in up to 100% of animals at week 20. Crescent formation began at week 10 and peaked at week 16, maximally affecting 57% of glomeruli. Crescent formation was initiated by "activated" podocytes that formed cell bridges between tuft and Bowman's capsule. The typical picture of a diffuse immune complex nephritis was found in all animals as early as 8 weeks. Fluorescence intensity increased with age and became strongly positive for immunoglobulin (Ig)A, IgM, IgG, and C3 in the mesangium and along the peripheral capillary loops. CONCLUSION: Although ANCAs were found in the majority of animals, the massive presence of glomerular immune deposits differed from the pauci-immune pattern found in human AASV, making this model not completely representative for human ANCA-associated CGN. However, the spontaneous and concomitant development of pANCA, small vessel vasculitis, and CGN raises the opportunity to analyze pathogenetic links between these disease manifestations in vivo.