Rate Of Outgrowth Research Articles

Effectiveness of passivation techniques on hydrogen desorption in a tritium environment

Tritium is a radioactive isotope of hydrogen. It is used as a fuel in fusion reactors, a booster material in nuclear weapons, and as a light source in commercial applications. When tritium is used in fusion reactors, and especially when used in the manufacture of nuclear weapons, purity is critical. For U.S. Department of Energy use, tritium is recycled by the Savannah River Site in South Carolina and is processed to a minimum purity of 99.5%. For use elsewhere in the country, it must be shipped and stored, while maintaining the highest purity possible. As an isotope of hydrogen, it exchanges easily with the most common isotope of hydrogen, protium. Stainless steel bottles are used to transport and store tritium. Protium, present in air, becomes associated in and on the surface of stainless steel during and after the manufacture of the steel. When filled, the tritium within the bottle exchanges with the protium in and on the surface of the stainless steel, slowly contaminating the pure tritium with protium. The stainless steel is therefore passivated to minimize the protium outgrowth of the bottles into the pure tritium. This research is to determine how effective different passivation techniques are in minimizing the contamination of tritium with protium. Additionally, this research will attempt to determine a relationship between surface chemistry of passivated steels and protium contamination of tritium. The conclusions of this research found that passivated bottles by two companies, which routinely provide passivated materials to the U.S. Department of Energy, provide low levels of protium outgrowth into pure tritium. A bottle passivated with a material to prevent excessive corrosion in a highly corrosive environment, and a clean and polished bottle provided outgrowth rates roughly twice those of the passivated bottles above. Beyond generally high levels of chromium, oxygen, iron, and nickel in the passivated bottles, there did not appear to be a strong correlation between surface chemistry in the surface of the bottles and protium outgrowth rates.

Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: Phenotype and transcriptome analysis

Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of Bacillus cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth were assessed at pH 5.5 without and with 0.75, 1.5 and 3.0 mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from reduced germination and outgrowth rates to complete blockage of germination at 3.0 mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5 mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multidrug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed.

Abstract 1358: Focal adhesion kinase (FAK) regulates myeloid lineage cell activities that control murine tumor progression

Abstract The tumor microenvironment is a critical regulator of tumor progression. Results from a broad spectrum of experimental models and analyses of human tumor specimens provide strong evidence that cells of the myeloid lineage have functional attributes that can potentiate the growth, invasion, and metastasis of cancer. Our approach to investigate the role of the microenvironment in tumor progression is centered on the activity of focal adhesion kinase (FAK) in myeloid lineage cells. We have recently shown in macrophages that FAK, a non-receptor tyrosine kinase, regulates adhesion dynamics and migration toward soluble growth factors and chemokines located at sites of tumor growth. Additionally, mice that have FAK conditionally deleted in this lineage display reduced peritoneal infiltration of macrophages following thioglycollate-induced inflammation. These data suggest that FAK may be involved in regulating the migration of myeloid lineage cells to the tumor and the response of macrophages to factors within the tumor microenvironment. We hypothesize that FAK regulates critical aspects of myeloid/macrophage behavior that allow these cells to support tumor progression and metastasis. This hypothesis is being tested in several tumor models. First, using myeloid-specific conditional FAK knockout mice (FAK-/-), we have tested this hypothesis in the context of both a xenograft tumor model using MDA-MB-231 breast cancer cells and a spontaneous breast tumor model driven by mammary-specific expression of the polyoma virus middle T antigen (MMTV-PyVmT). Significantly fewer tumors developed from orthotopic injection of MDA-MB-231 cells in mice lacking FAK in myeloid lineage cells. F4/80 staining of tumor sections from these mice showed reduced infiltration of macrophages into the tumor mass compared to tumors from wild type (WT) mice. Initial data from the spontaneous MMTV-PyVmT tumor model show no significant differences in time to tumor onset and rate of tumor outgrowth between the FAK-/- and WT mice. Analysis of the lung metastatic tumor burden is ongoing. Second, we have conducted preliminary experiments testing the influence of FAK expression in myeloid lineage cells on the adaptive immune response to tumor growth. Following subcutaneous implantation of the syngeneic B16/F1 melanoma cell line, we observed a significant delay in the onset of tumor formation in FAK-/- mice compared to WT mice. However, when a similar study was performed in a severe combined immunodeficient (SCID) background, tumor outgrowth was identical in WT and conditional knockout mice. Based on these results, we are currently investigating mechanisms through which FAK expression in myeloid lineage cells may regulate the response of lymphocytes to the early stages of tumor outgrowth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1358.

Re-epithelialization from human skin explant cultures is promoted by ligand-activated HER3 receptor

Ligand-stimulated epidermal growth factor receptor (EGFR/HER1) plays a fundamental role in skin biology as potent transducer of mitotic and anti-apoptotic stimuli in keratinocytes. In human epidermis, at least two additional EGFR family members--HER2 and HER3--are expressed but their biological functions in normal and diseased human skin remain obscure. Here, we studied the expression and biological impact of HER3 in regenerating human epidermis formed from skin explants adhered to acellular dermis. Neoepidermal HER3 expression was examined by quantitative real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot analysis. The dynamic effect of HER3 receptor stimulation by recombinant heregulin (HRG)-beta1 was assessed by fluorescence imaging of re-epithelialization. In the neoepidermis, HER3 mRNA and protein were detected with activated receptors being immunolocalized at basal and low suprabasal levels. Exogenous HRG-beta1 at 10-20 ng/ml increased the outgrowth rate corresponding to approximately 30% the response of exogenous EGF. The growth-promoting effect of HRG-beta1 was associated with enhanced HER3 phosphorylation, keratinocyte proliferation and thickening of viable neoepidermis whereas blockade of ligand-binding to HER3 delayed the outgrowth process and inhibited both constitutive and ligand-induced HER3 phosphorylation. HER2 antagonism using an anti-dimerization antibody, pertuzumab, impeded the re-epithelialization rate. In addition, a selective HER2 kinase inhibitor, CP654577, downregulated phospho-HER3 expression suggesting that transactivation of kinase-deficient HER3 was accomplished through dimerization with HER2. The study emphasizes the central role of EGFR in epidermal renewal and demonstrates that HRG-activated HER3 contributes to the outgrowth process of epidermis in vitro.

Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2™ cells using automated high-content image analysis

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling neurite outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of hN2™ human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24 h post-plating hN2™ cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, β III-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2™ cells proceeded rapidly, with a majority of cells extending one to three neurites by 48 h in culture. In addition, concentration-dependent decreases in neurite outgrowth and ATP-content were observed following treatment of hN2™ cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity of hN2™ cells to neurite outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2™ cells provide a model in which to investigate chemical effects on neurite outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines.

Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies

IntroductionMost human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated β-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth.MethodsPre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or γ-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated β-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses.ResultsCultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated β-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism.ConclusionsOur studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential.

Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

JLP (JNK-associated leucine zipper protein) is a novel scaffolding protein involved in JNK signaling. Although it is known that JLP is highly expressed in brain, the biological function of JLP in neuronal systems remains unknown. Here, we report a novel interaction between JLP and SCG10 (superior cervical ganglia clone 10), which is a microtubule-destabilizing factor that is essential for neurite outgrowth. Inhibition of endogenous JLP expression using small interference RNA methodology strongly enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Our results show that JLP negatively regulates NGF-induced neurite outgrowth by decreasing the level of phosphorylated SCG10. Furthermore, inhibition of JNK phosphorylation by a small molecule inhibitor, SP600125, resulted in inhibition of SCG10 phosphorylation and inhibition of neurite growth. Taken together, our results suggest that JLP negatively regulates NGF-induced neurite outgrowth through a sequestering mechanism that results in an attenuation of NGF-induced SCG10 phosphorylation.

Combined microfluidics/protein patterning platform for pharmacological interrogation of axon pathfinding

Assembly of functional neural circuits relies on the ability of axons to navigate a complex landscape of guidance cues in the extracellular environment. In this report, we investigate localized cell signaling in response to these cues by combining a microfabricated compartmentalization chamber with multicomponent, protein-micropatterned surfaces; this system offers improved spatial resolution and new capabilities for targeted manipulation of neuronal axons. We illustrate the potential of this system by addressing the role of fibroblast growth factor receptor (FGFR) signaling in modulating axon guidance by N-cadherin. Motor neurons that were derived from embryonic stem cells extend axons from one compartment through a microchannel barrier and into a second compartment containing patterns of N-cadherin, against a background of laminin. N-cadherin was effective in both guiding and accelerating motor axon outgrowth. Using the chamber system to target the application of pharmacological agents to specific parts of the neuron, we demonstrate that FGFR signaling in the axon but not the cell body increases the rate of axon outgrowth while not affecting guidance along N-cadherin. These results demonstrate that cell signaling must take into account the spatial layout of the cell. This new platform provides a powerful tool for understanding such effects over a wide range of signaling systems.

Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications

Cryopreserved amniotic membrane (Cryo-AM) is widely used in ocular surface surgery because of its positive effect on wound healing and its anti-inflammatory properties. A new peracetic acid/ethanol sterilized air-dried amniotic membrane (AD-AM) recently became available which might be an alternative to Cryo-AM. Our aim was to compare AM preserved with both methods with regard to the release of wound-healing modulating proteins, the preservation of basement membrane components, and the ability to serve as a substrate for the cultivation of human limbal epithelial cells (HLECs). Pieces of Cryo-AM and AD-AM from three different donors were incubated in DMEM for five days. The culture supernatant was collected after an incubation period of 0.1, 24, 48, 72 and 120 h; in the case of AD-AM, this period was extended up to 14 days. TIMP-1, IL-1ra, CTGF and TGF-beta1 were detected in the culture supernatant using Western blotting. Twenty human limbal epithelial cultures were initiated on both AD- and Cryo-AM. The cultures were analyzed morphologically, and the outgrowth area was measured in 3-day intervals. Cryosections of Cryo- and AD-AM from three different donors were analyzed histochemically to detect the basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin. The release of TIMP-1, IL-1ra and TGF-beta1 from Cryo-AM was constant for the studied period. CTGF showed a stronger signal after 120 h. None of the analyzed proteins, except for a small amount of IL-1ra, could be detected in the supernatant of AD-AM. An outgrowth of HLEC was observed in all cultures on Cryo-AM, but in only 30% of cultures on AD-AM. The outgrowth area on Cryo-AM was at all time points significantly higher than on AD-AM (p < 0.0001). Collagen IV, -VII, laminins and fibronectin were detectable in the basement membrane of Cryo-AM, but only collagen IV and fibronectin in AD-AM. Cryo-AM is a more suitable substrate for the cultivation of HLECs than AD-AM. The higher outgrowth rate of cultured limbal epithelium, release of intact soluble wound-healing modulating factors and a better preservation of basement membrane components suggest the superiority of Cryo-AM for use in ophthalmology in comparison to AD-AM.

Optical neuronal guidance in three-dimensional matrices

We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.

Quantifying Neurite Growth Mediated by Interactions among Secretory Vesicles, Microtubules, and Actin Networks

Neurite growth is a fundamental process of neuronal development, which requires both membrane expansions by exocytosis and cytoskeletal dynamics. However, the specific contribution of these processes has not been yet assessed quantitatively. To study and quantify the growth process, we construct a biophysical model in which we relate the overall neurite outgrowth rate to the vesicle dynamics. By considering the complex motion of vesicles in the cell soma, we demonstrate from biophysical consideration that the main step of finding the neurite initiation site relies mainly on a two-dimensional diffusion/sequestration/fusion at the cell surface and we obtain a novel formula for the flux of vesicles at the neurite base. In the absence of microtubules, we show that a nascent neurite initiated by vesicular delivery can only reach a small length. By adding the microtubule dynamics to the secretory pathway and using stochastic analysis and simulations, we study the complex dynamics of neurite growth. Within this model, depending on the coupling parameter between the microtubules and the neurite, we find different regimes of growth, which describe dendritic and axonal growth. To validate one aspect of our model, we demonstrate that the experimental flux of TI-VAMP but not Synaptobrevin 2 vesicles contributes to the neurite growth. We conclude that although vesicles can be generated randomly in the cell body, the search for the neurite position using the microtubule network and diffusion is quite fast. Furthermore, when the TI-VAMP vesicular flow is large enough, the interactions between the microtubule bundle and the neurite control the growth process. In addition, all of these processes intimately cooperate to mediate the various modes of neurite growth: the model predicts three different growing modes including, in addition to the stable axonal growth and the stochastic dendritic growth, a fast oscillatory regime. Finally our study demonstrates that cytoskeletal dynamics is necessary to generate long protrusion, while vesicular delivery alone can only generate small neurite.

Mitochondrial remodeling in differentiating neuroblasts

Mitochondria are able to change their shape through fission and fusion events, leading to a continuous remodeling of the mitochondrial network. Whereas the mitochondrial fission has been extensively studied and primarily related to the onset and progression of apoptosis, the physiological function of the opposite process of fusion is far less understood. With this study we analyzed the process of mitochondrial fusion in immortalized hippocampal neuroblasts searching for a relationship with specific changes in cellular physiology. The mitochondrial dynamics was examined in every stage of the cell cycle and a link was found between the enhancement of the mitochondrial transmembrane potential Δ Ψ m, the widespread mitochondrial fusion and the process of neurite outgrowth. An identical mitochondrial reorganization also appeared in response to the pro-differentiating agent retinoic acid. The single-cell analysis in time-lapse of the mitochondrial response to RA evidenced a free calcium raise in the mitochondrial matrix coupled with the Δ Ψ m increase and it confirmed the close coordination between these two events and the fusion of mitochondria. The modulation of oxidative phosphorylation by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or pyruvate, underscored the importance of Δ Ψ m changes both in shaping the mitochondrial network and in regulating the rate of neurite outgrowth. We also report that the mitochondrial fusion observed during neurite outgrowth is not a consequence of the microtubule reorganization, since pharmacological treatments capable of blocking the microtubule dynamics failed to inhibit the mitochondrial remodeling in response to RA.

The Effects of Cell Surface NgCAM Expression on Developing Chick Brain Axons In Vitro

During embryonic brain development, molecular guidance cues work in concert to transduce signals to guide axons to their appropriate targets. Of these cues are proteins of the Ig superfamily, including L1 and its chick homologue, neuron‐glia cell adhesion molecule (NgCAM). L1 and NgCAM are neuronal cell surface membrane proteins shown by others to accelerate axonogenesis and increase axon elongation. In vitro bioassays were performed in order to test the hypothesis that NgCAM expression on the cell monolayer will increase rate of outgrowth, axon length, and number of axon processes. Embryonic Day 7 optic tectum (OT) explants were first used to study outgrowth, yet failed to be an efficient and reproducible model. Aggregates were then made from dissociated OT cells and plated onto NIH3T3 monolayers that did or did not express uniform NgCAM and onto monolayers exhibiting patterned NgCAM expression. The aggregates were visualized using static images and time‐lapse microscopy by fluorescent membrane dye. Current data has shown an increase in both axon length and number in the presence of NgCAM, suggesting its crucial role as a positive axon guidance cue. Supported in part by HHMI and NIH.

Implantation of Schwann cells in rat tendon autografts as a model for peripheral nerve repair: Long term effects on functional recovery

Cultured Schwann cells in tendon autografts for nerve repair improve the early phase of nerve regeneration in rat sciatic nerves as judged by the rate of axonal outgrowth. We tested the long-term effects on functional recovery using measurements of muscle force, the number of axons and myelination, using morphometry. In addition, we recorded wet weight of the gastrocnemius muscle. Schwann cell cultures were prepared from predegenerated nerves. Ten and 15 mm defects in rat sciatic nerves were bridged using bilateral tendon autografts with Schwann cell-seeded tendon autografts on one side, and untreated tendon autografts on the other. Animals were evaluated at six and 12 weeks, respectively. At six weeks, myelination, as judged by G-ratio (ratio of axonal diameter to diameter of nerve fibres), was significantly increased in tendon autografts pretreated with Schwann cells in 10mm defects. No such difference was seen in the 15 mm defects. We found no difference in functional recovery, other morphometric variables, or muscle weight between the two grafts. We conclude that early effects on nerve regeneration using transplantation of cultured Schwann cells in rat sciatic nerves are temporary. Other strategies are necessary to obtain lasting effects on functional recovery.

Optimal Micropattern Dimensions Enhance Neurite Outgrowth Rates, Lengths, and Orientations

Micropattern dimensions can significantly influence neurite outgrowth orientation, rate, and length. Laminin micropatterns of various widths from 10 to 50 microm at 10 microm intervals separated by 40 microm spaces were generated on poly(methyl methacrylate) surfaces using microscale plasma-initiated patterning (microPIP). Dissociated dorsal root ganglion (DRG) neurons were seeded on the micropatterned surfaces and cultured for 24 h in serum-free media. Neurite outgrowth numbers, lengths, rates, and orientations were measured on all micropatterned substrates. The results indicated that the dimension of the laminin pattern influenced the neurite outgrowth length, rate, and orientation, but not the numbers of neurite outgrowth. Neurons on more than 30 microm wide laminin pattern showed faster neurite outgrowth compared to other dimensions, and relatively low orientation at 50 microm pattern dimensions. Neurites at 40 microm laminin pattern widths demonstrated the fastest outgrowth rates and were highly oriented. The 40 microm laminin dimension is wide enough to provide sufficient laminin amounts for neuron growth and narrow enough to efficiently guide neurites. Based on these results, adhesive protein micropatterns of 40 microm dimensions are recommended when investigating DRG neurons.

Cumulative activation of axillary buds by nodal roots in Trifolium repens L.

In Trifolium repens the rate of outgrowth of an axillary bud was closely correlated with its duration of exposure to a nearby nodal root. The dose-dependent nature of this relationship, over 0-22 d, is consistent with the concept that axillary buds are cumulatively activated by a root signal (RS) such that the longer they receive the signal the higher is their level of activation and hence their rate of outgrowth. Furthermore, the activation level attained by a bud was subsequently retained following the excision of the nodal root providing the source of its activation: its rate of growth 3-6 weeks after root excision still reflected the initial level of activation of the bud. Thus, once activated, a bud required relatively little RS to maintain its rate of outgrowth, implying that activation involves the establishment of an autonomous control mechanism within the bud itself. This provides an explanation of how a strongly activated apical bud can continue growth at relatively low RS levels when it is distanced from its nearest root system, while at the same time the prevailing low RS environment leads to weak activation of the axillary buds emerging from it.

Arabidopsis Inositol Polyphosphate 6-/3-Kinase (AtIpk2β) Is Involved in Axillary Shoot Branching via Auxin Signaling

The Arabidopsis (Arabidopsis thaliana) inositol polyphosphate 6-/3-kinase gene (AtIpk2beta) is known to participate in inositol phosphate metabolism. However, little is known about its physiological functions in higher plants. Here, we report that AtIpk2beta regulates Arabidopsis axillary shoot branching. By overexpressing AtIpk2beta in the wild type and mutants, we found that overexpression of AtIpk2beta leads to more axillary shoot branches. Further analysis of AtIpk2beta overexpression lines showed that axillary meristem forms earlier and the bud outgrowth rate is also accelerated, resulting in more axillary shoot branches. The AtIpk2beta promoter/beta-glucuronidase (GUS) fusion (AtIpk2betaGUS) expression pattern is similar to that of the auxin reporter DR5GUS. Moreover, AtIpk2beta can be induced in response to exogenous indole-3-acetic acid (IAA) treatments. In addition, AtIpk2beta overexpression plants exhibit IAA-related phenotypes and are more resistant to exogenous IAA treatments. Further analysis employing reverse transcription-polymerase chain reaction shows that some genes, including auxin-biosynthesis (CYP83B1), auxin-transport (PIN4), and auxin-mediated branching genes (MAX4 and SPS), are regulated by AtIpk2beta. Taken together, our data provide insights into a role for AtIpk2beta in axillary shoot branching through the auxin signaling pathway.

Rate of neurite outgrowth in sympathetic neurons is highly resistant to suppression of protein synthesis: Role of protein degradation/synthesis coupling

Neurites projecting to their target tissues during embryogenesis are subject to many perturbations that could influence their rate of growth. For example, environmental influences such as supply of neurotrophic factor or electrical activity profoundly influence the rate of neuronal protein synthesis. Because accumulation of protein is necessary for outgrowth to proceed normally, a perturbation in protein synthesis could cause a net change in the rate of accumulation of proteins with the result that neurite outgrowth rate increases or decreases. That neurite outgrowth does not normally seem to be subject to such perturbations suggests involvement of a homeostatic system controlling the rate of outgrowth. Consistent with this hypothesis, we show here that the rate of growth of neurites of sympathetic neurons is highly resistant to decreased rates of protein synthesis. Chronic suppression of protein synthesis by 60% had no significant effect on neurite outgrowth over a 2-day period while complete suppression halted it almost immediately. By the 3rd day of exposure, 60% suppression slowed outgrowth. Sustained suppression of protein synthesis rate by 33% had no effect on rate of outgrowth even after 7 days. We show that the ability of the growing neurites to resist protein synthesis suppression appears to be caused, at least in part, by a parallel decrease in the rate of protein degradation. The result of this coupling between degradation and synthesis is that proteins can continue to accumulate even when protein synthesis rate decreases, allowing normal rates of neurite outgrowth.

IGF-I specifically enhances axon outgrowth of corticospinal motor neurons

Corticospinal motor neurons (CSMN) are among the most complex CNS neurons; they control voluntary motor function and are prototypical projection neurons. In amyotrophic lateral sclerosis (ALS), both spinal motor neurons and CSMN degenerate; their damage contributes centrally to the loss of motor function in spinal cord injury. Direct investigation of CSMN is severely limited by inaccessibility in the heterogeneous cortex. Here, using new CSMN purification and culture approaches, and in vivo analyses, we report that insulin-like growth factor-1 (IGF-I) specifically enhances the extent and rate of murine CSMN axon outgrowth, mediated via the IGF-I receptor and downstream signaling pathways; this is distinct from IGF-I support of neuronal survival. In contrast, brain-derived neurotrophic factor (BDNF) enhances branching and arborization, but not axon outgrowth. These experiments define specific controls over directed differentiation of CSMN, indicate a distinct role of IGF-I in CSMN axon outgrowth during development, and might enable control over CSMN derived from neural precursors.

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model

We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and "purse string"-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent "purse string"-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype.