Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors.

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.     

Characterization and purification of polymorphic arylalkylamine N-acetyltransferase from the American cockroach, Periplaneta americana.

We separated two forms of arylalkylamine N-acetyltransferase (AANAT) from various organs of the American cockroach, Periplaneta americana. Both forms of the enzyme had an equivalent molecular mass of 28 kDa. One form isolated from the testicular accessory glands had high enzyme activity at acidic pHs. The isoelectric point was 5-6 and the substrate specificity was wider than the other type. The other isolated form from female midguts had a higher level of enzyme activity at basic pHs. These findings suggested that P. americana contains polymorphic AANAT, as is the case in Drosophila melanogaster. These forms differed not only in pH specificity, and substrate specificity but in chromatographic behavior and kinetic properties. Most of the organs we examined contained a mixture of the two forms since two types of AANAT activity were separated in different chromatographic fractions when two pH conditions were used for activity measurement.     

Fibroblast growth factor signaling in Caenorhabditis elegans.

Growth factor receptor tyrosine kinases (RTKs), such as the fibroblast growth factor receptor (FGFR), play a major role in how cells communicate with their environment. FGFR signaling is crucial for normal development, and its misregulation in humans has been linked to developmental abnormalities and cancer. The precise molecular mechanisms by which FGFRs transduce extracellular signals to effect specific biologic responses is an area of intense research. Genetic analyses in model organisms have played a central role in our evolving understanding of these signal transduction cascades. Genetic studies in the nematode C. elegans have contributed to our knowledge of FGFR signaling by identifying genes involved in FGFR signal transduction and linking their gene products together into signaling modules. This review will describe FGFR-mediated signal transduction in C. elegans and focus on how these studies have contributed to our understanding of how FGFRs orchestrate the assembly of intracellular signaling pathways.     

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.     

Three-dimensional geometric morphometrics of Arvicanthis: implications for systematics and taxonomy

Arvicanthis is an African murid, found throughout sub-Saharan Africa, Sudan and Egypt. Although in the past 10 years several studies have been carried out to assess its systematics, there is still a need for a general revision of the genus. In this study the morphometric relationships between 71 populations throughout the range were investigated. A three-dimensional geometric morphometric approach was used to assess differences in the size and shape of the skull. These were related to the different biogeographical domains characterizing the range of the genus and to molecular and karyotypic phylogenies. Results agree only in part with phylogeny, and show a close relationship with the environmental backgrounds of each species. It is therefore suggested that the adaptation of Arvicanthis to local environment has played an important role in the phenotypic evolution of the skull. This leads to problems in taxonomic definitions based on morphometrics, which should not be used without comparison with other independently derived characters such as the DNA and the karyotype.     

Regulation of Apterous activity in Drosophila wing development.

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Here, we present evidence that complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. We also report that a difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo.     

Populations of NGF-dependent neurones differ in their requirement for BAX to undergo apoptosis in the absence of NGF/TrkA signalling in vivo.

Reports that apoptosis within populations of neurotrophin-dependent neurones is virtually eliminated in BAX-deficient mice and that BAX-deficient neurones survive indefinitely in culture without neurotrophins have led to the view that BAX is required for the death of neurotrophin-deprived neurones. To further examine this assertion in vivo, we have studied two populations of NGF-dependent neurones during the period of naturally occurring neuronal death in mice that lack BAX, NGF or the NGF receptor TrkA, alone and in combination. In the superior cervical ganglion (SCG), naturally occurring neuronal death and the massive loss of neurones that took place in the absence of NGF or TrkA were completely prevented by elimination of BAX. However, in the trigeminal ganglion, naturally occurring neuronal death was only partly abrogated by the elimination of BAX, and although the massive neuronal death that took place in this ganglion in the absence of NGF or TrkA was initially delayed in embryos lacking BAX, this subsequently occurred unabated. Accordingly, BAX-deficient neurones survived in defined without NGF whereas BAX-deficient trigeminal neurones died in the absence of NGF. These results indicate that whereas BAX is required for the death of SCG neurones during normal development and when these neurones are deprived of NGF/TrkA signalling in vivo, the death of trigeminal ganglion neurones occurs independently of BAX when they are deprived of NGF/TrkA signalling. We conclude that BAX is not universally required for neuronal death induced by neurotrophin deprivation, but that there are major differences for the requirement for BAX among different populations of NGF-dependent neurones.     

PYCNOGENOL chewing gum minimizes gingival bleeding and plaque formation.

PYCNOGENOL is an antioxidant phytochemical shown to have antiinflammatory activity in both the in vitro and in vivo models. This study compared the effects of chewing gums with and without PYCNOGENOL on gingival bleeding and plaque formation in 40 human subjects. In this double-blind study, subjects were assigned randomly to receive either control gums without PYCNOGENOL or experimental gums containng 5 mg PYCNOGENOL. Subjects used chewing gums for 14 days. Gingival bleeding and plaque scores were taken before and after the experiment. PYCNOGENOL chewing gums significantly reduced gingival bleeding, while no changes were noted in bleeding indexes in control subjects who used regular chewing gums. Subjects using regular control gums had significant increases of dental plaque accumulation during the two-week period. No increases in plaque accumulation were noted in subjects using PYCNOGENOL chewing gums. The data of this study suggest that the use of Pycnogenol chewing gums can minimize gingival bleeding and plaque accumulation.     

The exosome of Trypanosoma brucei.

The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species. An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.