We used a kinematic analysis to investigate the growth processes responsible for variation in primary root growth between 18 ecotypes of Arabidopsis. Root elongation rate differed 4-fold between the slowest (Landsberg erecta, 71 m h Ϫ1 ) and fastest growing line (Wassilewskija [Ws]; 338 m h Ϫ1 ). This difference was contributed almost equally by variations in mature cortical cell length (84 [Ws]). We found no correlation between mature cell size and endoreduplication, refuting the hypothesis that the two are linked. However, there was a strong correlation between cell production rates and the activity of the cyclin-dependent kinase (CDKA). The level of the protein could explain 32% of the variation in CDKA. Therefore, it is likely that regulators of CDKA, such as cyclins and inhibitors, are also involved. These data provide a functional link between cell cycle regulation and whole-plant growth rate as affected by genetic differences.The rate at which plants grow is an important agronomic trait in cultivated plants, as well as an adaptive trait under natural growth conditions. Therefore, the physiological characteristics associated with fast and slow growth have been extensively investigated (for an overview, see Lambers et al., 1998). They involve the acquisition of growthsupporting substances (photosynthesis and nutrient uptake) and their utilization (anatomy, chemical composition, cell division, and cell expansion). In contrast to the multitude of physiological investigations into the basis of growth rate differences, genetic studies are scarce. Arabidopsis, the model plant for genetic research, has been collected from a wide range of habitats distributed primarily over most of the northern hemisphere. Genetic differences between local populations (commonly called ecotypes, despite that this term does not conform strictly to its ecological definition [Pigliucci, 1998]) are presumably associated with adaptation to the prevailing environmental conditions. Numerous ecotypes were collected by the pioneers of Arabidopsis research and have since been use to investigate a wide range of physiological processes, through comparisons between ecotypes and by genetic mapping using recombinant inbred lines (Alonso-Blanco and Koornneef, 2000). With some notable exceptions (Li et al., 1998), little is published about differences in the rate at which Arabidopsis ecotypes grow when compared under standardized laboratory conditions.There are two complementary views on how root growth is regulated (Silk, 1984). The spatial model describes at what rate division and expansion occur as a function of position along the root axis. Root elongation rate in this model is determined by the size of the growth zone and local rates of expansion. According to this view, cell division merely subdivides cellular space provided by the expansion process. In an alternate manner, a cellular model can be adopted whereby cell production in the meristem drives growth by producing the cells that will subsequently expand to reach a given mature size ...

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an ␣-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and ␤-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.Most protein-based therapeutic agents produced in heterologous expression systems are glycosylated, a modification that is crucial for correct folding, stability, and bioactivity of the protein and influences its pharmacokinetic properties, such as tissue distribution and blood clearance. Glycoproteins with terminal sialic acids on their glycans persist longer in the blood than glycoproteins with terminal galactose, N-acetylglycosamine, or mannose residues because the latter compounds are cleared rapidly via receptors in the liver and on reticuloendothelial cells (e.g., the asialoglycoprotein receptor and the mannose receptor) (10,20,30,31). In addition, glycan structures produced in nonhuman cells can cause immune reactions, as exemplified by the reaction against xenografts of porcine origin; these reactions are primarily caused by the presence of ␣-galactose on the glycoproteins (7). Another example is the immune reaction against glycoproteins from yeast, which results from the presence of ␣-1,3-mannose, ␤-linked mannose, and/or phosphate residues in either a phosphomonoester or phosphodiester linkage (1, 32). Consequently, recombinant glycoproteins produced for therapeutic applications should be expressed in heterologous hosts that produce protein-linked oligosaccharides that closely resemble those of humans.

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A direct link between receptor glycosylation and activation following natural ligand interaction has not been observed. Here, we discover a membrane sialidase-controlling mechanism that depends on ligand binding to its receptor to induce enzyme activity which targets and desialylates the receptor and, consequently, causes the induction of receptor dimerization and activation. We also identify a specific sialyl alpha-2,3-linked beta-galactosyl sugar residue of TrkA tyrosine kinase receptor, which is rapidly targeted and hydrolyzed by the sialidase. Trk-expressing cells and primary cortical neurons following stimulation with specific neurotrophic growth factors express a vigorous membrane sialidase activity. Neuraminidase inhibitors, Tamiflu, BCX1812, and BCX1827, block sialidase activity induced by nerve growth factor (NGF) in TrkA-PC12 cells and by brain-derived neurotrophic factor (BDNF) in primary cortical neurons. In contrast, the neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, specific for plasma membrane ganglioside Neu3 and Neu2 sialidases has no inhibitory effect on NGF-induced pTrkA. The GM1 ganglioside specific cholera toxin subunit B applied to TrkA-PC12 cells has no inhibitory effect on NGF-induced sialidase activity. Neurite outgrowths induced by NGF-treated TrkA-PC12 and BDNF-treated PC12(nnr5) stably transfected with TrkB receptors (TrkB-nnr5) cells are significantly inhibited by Tamiflu. Our results establish a novel mode of regulation of receptor activation by its natural ligand and define a new function for cellular sialidases.