Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

Bioreactor expansion of human neural precursor cells in serum‐free media retains neurogenic potential

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinson's disease (PD), Huntington's disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum‐free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF‐h2) for the expansion of hNPCs, achieving an overall cell‐fold expansion of 10¹³ over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled‐up from stationary culture to suspension bioreactors using this medium. Serial subculturing of the cells in suspension bioreactors resulted in an overall cell‐fold expansion of 7.8 × 10¹³ after 140 days. These expanded cells maintained their multipotency including the capacity to generate large numbers of neurons (about 60%). In view of our previous studies regarding successful transplantation of the bioreactor‐expanded hNPCs in animal models of neurological disorders, these results have demonstrated that PPRF‐h2 (containing dehydroepiandrosterone, basic fibroblast growth factor and human leukemia inhibitory factor) can successfully facilitate the production of large quantities of hNPCs with potential to be used in the treatment of neurodegenerative disorders. Biotechnol. Bioeng. 2010. 105: 823–833. © 2009 Wiley Periodicals, Inc.     

High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia alpha2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.     

Kinetic and mechanistic considerations in the gelation of genipin-crosslinked gelatin

The gelling properties (gel time (t_gel) and gel strength) of a 10% (w/w) gelatin sol were investigated as a function of genipin (GP) concentration (0–15 mM) and temperature (25–55 °C) to discern mechanisms and optimal conditions for fixation. Gel time increased with increasing temperature, reached a maximum, and then declined as temperature was raised further. By contrast, network strength data followed the opposite trend. From the thermal behavior of t_gel and network strength, it was inferred that gelation in the low-temperature regime was dominated by hydrogen bonding, while in the high-temperature regime it was dominated by covalent crosslinking. At higher temperatures, crosslinking was described by an Arrhenius rate law expression, with activation energies between 63.2 and 67.8 kJ/mol, depending on GP concentration. In the low temperature regime, an Arrhenius plot resulted in negative activation energies of −75.8 and −64.4 kJ/mol in the presence of 10 and 15 mM GP, respectively. With an increase in both GP concentration and temperature, the gelatin network gradually shifted from being dominated by hydrogen bonds (physical crosslinks) to covalent crosslinking (chemical crosslinks).     

Some physical and microstructural properties of genipin-crosslinked gelatin-maltodextrin hydrogels

The physical properties and microstructure of gelatin-maltodextrin hydrogels fixed with genipin (GP) were investigated as a function of pH (3-7), maltodextrin (MD) (0-9%, w/w) and GP (0-10 mM levels), at a constant gelatin (G) concentration (10%, w/w). Network strength (elastic modulus, E) and swelling behavior were characterized by large deformation testing and by swelling index (SI). In general, network strength increased and swelling decreased at higher pH, MD and GP levels, except at pH 3, where E was independent of the GP concentration until approximately 7.5 mM, above which it declined. Confocal scanning laser microscopy (CLSM) images showed phase separation to be suppressed at pH 3, whereas at pH 7, separation into a self-similar dispersed phase was apparent. Overall, the judicious use of GP to crosslink G was an appropriate means of kinetically trapping MD within the gelatin network.     

FABP3 and FABP10 in Atlantic salmon (Salmo salar L.)--general effects of dietary fatty acid composition and life cycle variations

The increased use of dietary plant oil supplementation combined with high dietary lipid loads challenges the lipid transport systems of cultivated fish species. Fatty acid binding proteins (FABPs) have been thoroughly studied as intracellular fatty acid transporters in vertebrates, but no data have been reported in Atlantic salmon. In the present study, comparative characterizations were performed, and dietary influence of plant oil supplementation on FABP3 and FABP10 expression was studied for several tissues in two separate dietary trials. In trial I, groups (6 fish each) were fed diets for 42 weeks (body mass 142+/-1 to 1463+/-83 g) (mean+/-S.D.), containing graded levels of rapeseed oil substituting for fish oil using a linear regression design. In trial II, groups (3 fish each) were fed 100% fish oil or 100% plant oil for 22 months (0.160+/-0.052 to 2523+/-590 g) (mean+/-S.D.) and sampled at regular intervals. Liver and muscle tissues appeared to express several FABPs possibly linked to different metabolic functions. FABPs mRNA expression did not change with dietary inclusion of 75% rapeseed oil, whereas FABP3 protein expression seemed to be affected by dietary rapeseed oil inclusion. Significant changes in red muscle FABP3 mRNA expression correlate to significant changes in total beta-oxidation capacity during the energy consuming process of smoltification.     

Activity-dependent growth of new dendritic spines is regulated by the proteasome

Growth of new dendritic spines contributes to experience-dependent circuit plasticity in the cerebral cortex. Yet the signaling mechanisms leading to?new spine outgrowth remain poorly defined. Increasing evidence supports that the proteasome is an important mediator of activity-dependent neuronal signaling. We therefore tested the role of the proteasome in activity-dependent spinogenesis. Using pharmacological manipulations, glutamate uncaging, and two-photon imaging of GFP-transfected hippocampal pyramidal neurons, we demonstrate that acute inhibition of the proteasome blocks activity-induced spine outgrowth. Remarkably, mutation of serine 120 to alanine of the Rpt6 proteasomal subunit in individual neurons was sufficient to block activity-induced spine outgrowth. Signaling through NMDA receptors and CaMKII, but not PKA, is required to facilitate spine outgrowth. Moreover, abrogating CaMKII binding to the NMDA receptor abolished activity-induced spinogenesis. Our data support a model in which neural activity facilitates spine outgrowth via an NMDA receptor- and CaMKII-dependent increase in local proteasomal degradation.     

Mutual antagonism between IP3RII and miRNA-133a regulates calcium signals and cardiac hypertrophy

Inositol 1,4,5′-triphosphate receptor II (IP₃RII) calcium channel expression is increased in both hypertrophic failing human myocardium and experimentally induced models of the disease. The ectopic calcium released from these receptors induces pro-hypertrophic gene expression and may promote arrhythmias. Here, we show that IP₃RII expression was constitutively restrained by the muscle-specific miRNA, miR-133a. During the hypertrophic response to pressure overload or neurohormonal stimuli, miR-133a down-regulation permitted IP₃RII levels to increase, instigating pro-hypertrophic calcium signaling and concomitant pathological remodeling. Using a combination of in vivo and in vitro approaches, we demonstrated that IP₃-induced calcium release (IICR) initiated the hypertrophy-associated decrease in miR-133a. In this manner, hypertrophic stimuli that engage IICR set a feed-forward mechanism in motion whereby IICR decreased miR-133a expression, further augmenting IP₃RII levels and therefore pro-hypertrophic calcium release. Consequently, IICR can be considered as both an initiating event and a driving force for pathological remodeling.     

Subunit-specific modulation of [(3)H]MK-801 binding to NMDA receptors mediated by dopamine receptor ligands in rodent brain

Dopamine D1 receptor (D1R) ligands may directly interact with the NMDA receptor (NMDAR), but detailed knowledge about this effect is lacking. Here we identify D1R ligands that directly modulate NMDARs and examine the contributions of NR2A and NR2B subunits to these interactions. Binding of the open channel blocker [(3)H]MK-801 in membrane preparations from rat- and mouse brain was used as a biochemical measure of the functional state of the NMDAR channel. We show that both D1R agonist A-68930 and dopamine receptor D2 antagonist haloperidol can decrease [(3)H]MK-801 binding with increased potency in membranes from the NR2A(-/-) mice (i.e. in membranes containing NR2B only), as compared to the inhibition obtained in wild-type membranes. Further, a wide range of D1R agonists such as A-68930, SKF-83959, SKF-83822, SKF-38393 and dihydrexidine were able to decrease [(3)H]MK-801 binding, all showing half maximal inhibitory concentrations ~20 μM, and with significant effects occurring at or above 1 μM. With membranes from D1R(-/-) mice, we demonstrate that these effects occurred through a D1R-independent mechanism. Our results demonstrate that dopamine receptor ligands can selectively influence NR2B containing NMDARs, and we characterize direct inhibitory NMDAR effects by different D1R ligands.     

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.