Diffusion in axon guidance

Axon guidance by target-derived diffusible factors plays an important role in the development of the nervous system. This paper considers the constraints imposed on this process by the mathematics of diffusion. A point source continuously producing a factor into an infinite three-dimensional volume is considered as a model for both the in vivo and in vitro situation. Basic constraints for effective guidance are assumed to be that the concentration falls between certain maximum and minimum limits, and that the percentage change in concentration across the width of the growth cone exceeds a certain minimum value. The evolution of the shape of the gradient over time is analysed. Using biologically reasonable parameter values, it is shown that the maximum range over which growth cone guidance by a diffusible factor is possible for large times (several days) after the start of the production of the factor is 500-1000 microm. This maximum distance is independent of the diffusion constant of the diffusing molecule, applies to both chemoattractants and chemorepellents, and agrees with experimental data. At earlier times, however, the constraints may be satisfied for distances up to several millimetres. The time it takes for this maximum guidance distance to fall to the asymptotic value depends on the diffusion constant. This time is a few hours for a small molecule but as much as a few days for a large molecule. The model therefore predicts that guidance over distances larger than 1000 microm is possible if the start of production of the factor is carefully matched to the time when guidance is required.     

Tyrosine kinase inhibition prevents deformation-stimulated vascular smooth muscle growth

The goal of this study was to determine the role of tyrosine phosphorylation in transducing deformation-stimulated vascular smooth muscle growth. Rat aorta-derived vascular smooth muscle cells were cultured on flexible silicone elastomer membranes and subjected to cyclic deformation (15 cycles per minute, deformed 2 seconds, relaxed 2 seconds). Deformation significantly increased proto-oncogene expression, [3H]thymidine incorporation, [3H]leucine incorporation, and cell number. Time course studies showed an 8-hour lag between initiation of cell deformation and onset of [3H]thymidine incorporation, with peak levels achieved after 18 to 24 hours. Western analysis of protein blots from deformed cells (10 minutes) demonstrated increased levels of phosphotyrosine-containing proteins having molecular weights of 110 to 130 and 70 to 80 kD. Deformation-stimulated tyrosine phosphorylation was prevented by the tyrosine kinase inhibitor Herbimycin A. Tyrosine kinase inhibition also prevented deformation-stimulated vascular smooth muscle cell growth as measured by [3H]thymidine incorporation. Cyclic deformation stimulates vascular smooth muscle proliferation through activation of tyrosine kinases. Inhibition of tyrosine phosphorylation is an effective means of preventing deformation-induced vascular smooth muscle growth in vitro.     

Tyrosine kinase inhibition: an approach to drug development

Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.     

Genetic analysis on the role of integrin during axon guidance in Drosophila

Heterodimeric cell surface receptor integrin is widely expressed in the nervous system, but its specific role during axon development has not been directly tested in vivo. We show that the Drosophila nervous system expresses low levels of positron-specific (PS) integrin subunits alphaPS1, alphaPS2, and betaPS during embryonic axogenesis. Furthermore, certain subsets of neurons express higher levels of integrin mRNAs than do the rest. Null mutations in either the alphaPS1 or alphaPS2 subunit gene cause widespread axon pathfinding errors that can be rescued by supplying the wild-type integrin subunit to the mutant nervous system. In contrast, misexpressing either the alphaPS1 or alphaPS2 integrin subunit in all neurons leads to no obvious axon pathfinding errors. We propose that integrin does not itself serve as either a "clutch" constituting molecule or a specific growth cone "receptor," as proposed previously, but rather as part of a molecular network that cooperatively guarantees accurate axon guidance.     

Tyrosine kinase inhibitor herbimycin A reduces the stability of cyclin-dependent kinase Cdk6 protein in T-cells

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.     

Tyrosine kinase: implications in tumor pathology and therapeutic perspectives

PURPOSE: Both generalist and pulmonologist physicians care for patients with severe chronic obstructive pulmonary disease (COPD). We studied patients hospitalized with severe COPD to explore whether supervision of care by pulmonologists is associated with greater costs or better survival. SUBJECTS AND METHODS: We studied 866 adults with severe COPD enrolled in the Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatments (SUPPORT), a prospective study at five academic medical centers. Patients were admitted to the hospital or transferred to an intensive care setting for treatment of severe COPD, defined by hypoxia (PaO2 <60 mm Hg) and hypercapnia (PaCO2 >50 mm Hg) or hypercapnia alone if on supplemental oxygen. Resource intensity was measured using a modified version of the Therapeutic Intervention Scoring System and estimated hospital costs. To account for differences in the patient case mix, propensity scores were developed to represent each patient's probability of having a pulmonologist as attending physician and each patient's probability of being in an intensive care unit (ICU) at study admission. RESULTS: Of the 866 patients studied, 512 had generalists and 354 pulmonologists as their attending physicians. The median patient age was 70 years; 52% were male; 14% died within 30 days. After adjusting for baseline differences in patient characteristics, there were no differences in resource intensity and hospital costs in those treated by pulmonologists or generalists. Adjusted average resource intensity scores for the entire hospitalization were 16.5 for pulmonologists and 17.0 for generalists (P = 0.34). Estimated hospital costs were the same ($6,400) for patients treated by pulmonologists and generalists (P = 0.99). Patients with pulmonologists as attending physicians did not experience better survival. Comparing patients of pulmonologists to patients of generalists, the adjusted hazard ratio for 30-day mortality was 1.6 (95% confidence interval: 0.98, 2.5); the hazard ratio for 180-day mortality was 1.2 (0.9, 1.7). CONCLUSIONS: Our findings suggest that for patients hospitalized with exacerbation of severe COPD, those with pulmonologist attending physicians do not have higher hospital resource use or better survival than those with generalist attending physicians.     

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-beta and ori-gamma) being somewhat preferred. We show here that the chromatin in the ori-beta and ori-gamma regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.     

The transmembrane Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon guidance

Parasite-derived antioxidant proteins have been implicated in playing an important role in protection against the oxygen radicals that are generated during aerobic metabolism and in defense against host immune cell attack. Here we report that filarial nematodes include the thioredoxin peroxidase/thiol-specific antioxidant (TPx/TSA) family of antioxidant proteins as part of their complex defense against radical-mediated damage. At the protein level, the TPx/TSA from Brugia malayi (Bm-TPx-1) was approximately 50% identical and approximately 60% similar to TPx/TSAs from mammals, amphibians and yeast. Bm-TPx-1 was also approximately 60% identical to putative TPx proteins from a related filarial nematode, Onchocerca volvulus, and from the free-living nematode Caenorhabditis elegans. That B. malayi may express multiple forms of molecules with TPx/TSA activity was indicated by the identification of a B. malayi gene encoding a second, distinct member of the TPx/TSA family (Bm-tpx-2). Bm-tpx-1 was found to be transcribed in all stages of the parasite present in the mammalian host and the 25 kDa translation product was present in all of the developmental stages studied. The results of immunohistochemical, immunofluorescent and immunoprecipitation studies showed Bm-TPx-1 to be localized in the cells of the hypodermis/lateral chord in adult parasites and not to be present at the surface or in excretory/secretory products. The distribution in the parasite suggests that Bm-TPx-1 may play its major role in countering radicals produced within cells. A recombinant form of Bm-TPx-1 was biologically active and capable of protecting DNA from oxygen radical-mediated damage. Thioredoxin peroxidases may prove to be a critical component in the parasite's defense against injury caused by oxygen radicals derived from endogenous and exogenous sources.     

Developmental exposure to Aroclor 1254 produces low-frequency alterations in adult rat brainstem auditory evoked responses

Developmental exposure of Long-Evans rats to 0, 1, 4, or 8 mg/kg/day Aroclor 1254 (A1254) from Gestational Day 6 through Postnatal Day 21 produces an elevated behavioral threshold for a 1-kHz tone. Brainstem auditory evoked responses (BAERs) were assessed in a subset of these animals (about 1 year old) using filtered clicks at 1 (65 and 80 dB SPL), 4 (60 and 80 dB SPL), 16 (40 and 80 dB SPL), and 32 (40 and 80 dB SPL) kHz. Aroclor 1254 decreased BAER amplitudes at 1 and 4 kHz, but not at 16 or 32 kHz. A dose-related decrease in the baseline-to-peak P1A amplitude was observed for the 1-kHz (80-dB) stimulus. Doses of 1, 4, or 8 mg/kg/day A1254 decreased the peak-to-peak amplitude of both P1AN1 and P1BN1 for a 1-kHz (80-dB) stimulus. Doses of 4 and 8 mg/kg/day A1254 decreased the peak-to-peak amplitude of N1P2 and P2N2 for a 4-kHz (60-dB) or 1-kHz (80-dB) stimulus. At 8 mg/kg/day, A1254 also increased the latency of peak P4 at 1 kHz (65 dB). The decreases in peak P1A amplitudes are consistent with a dysfunction of the cochlea and/or auditory nerve. Together, the data confirm that developmental exposure of rats to A1254 produces a permanent low- to mid-frequency auditory dysfunction and suggest a cochlear and/or auditory nerve site of action.     

Neurolin Ig domain 2 participates in retinal axon guidance and Ig domains 1 and 3 in fasciculation

The optic disk-directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation (Ott, H., M. Bastmeyer, and C.A.O. Stuermer, 1998. J. Neurosci. 18:3363-3372). New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition molecule with five Ig domains, were generated to assign function (guidance versus fasciculation) to specific Ig domains. By their ability or failure to recognize Chinese hamster ovary cells expressing recombinant neurolin with deletions of defined Ig domains, mAbs were identified as being directed against Ig domains 1, 2, or 3, respectively. Repeated intraocular injections of a mAb against Ig domain 2 disturb the disk-directed growth: axons grow in aberrant routes and fail to reach the optic disk, but remain fasciculated. mAbs against Ig domains 1 and 3 disturb the formation of tight fascicles. mAb against Ig domain 2 significantly increases the incidence of growth cone departure from the disk-oriented fascicle track, while mAbs against Ig domains 1 and 3 do not. This was demonstrated by time-lapse videorecording of labeled growth cones. Thus, Ig domain 2 of neurolin is apparently essential for growth cone guidance towards the disk, presumably by being part of a receptor (or complex) for an axon guidance component.     

Role of a new Rho family member in cell migration and axon guidance in C. elegans

Rho family GTPases are thought to regulate actin-dependent processes, but their functions in vivo are still poorly understood. We have investigated the function of a new, widely expressed Rho family member in C. elegans by analyzing mutations in the endogenous gene. Activated and null alleles all inhibit cell migration, demonstrating that this protein is required for cell migration in vivo. Only a small subset of the migrations inhibited by activating mutations are inhibited by null mutations, suggesting that considerable functional redundancy exists within this system. Our findings support this conclusion and show that mig-2 functions redundantly with another pathway to regulate nuclear migration. Surprisingly, activated alleles also cause misguided axon growth, suggesting that Rho family GTPases may couple guidance cues to process outgrowth.     

Tyrosine phosphorylation of extracellular signal-regulated protein kinase 4 in response to growth factors

Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this re-agent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.     

Tyrosine phosphorylation of the muscle-specific kinase is exclusively induced by acetylcholine receptor-aggregating agrin fragments

During formation of the neuromuscular junction, the basal membrane protein agrin initiates the aggregation of acetylcholine receptors (AChR) on the surface of myotubes. A muscle-specific kinase (MuSK) becomes phosphorylated upon incubation with agrin, although it does not bind to agrin on its own. Utilizing MuSK-specific antibodies, we demonstrate that the ability of different splicing variants and truncation fragments of agrin to trigger MuSK phosphorylation and AChR aggregation are correlated. Only agrin forms which are potent inducers of AChR-clustering are able to trigger the phosphorylation of MuSK. Picomolar concentrations of agrin are already sufficient to induce MuSK phosphorylation. Similar amounts are necessary for the aggregation of AChRs as well as their phosphorylation on a tyrosine residue. The complete overlap of specificities for MuSK phosphorylation and AChR aggregation suggests that only binding of agrin to a MuSK-containing receptor complex is responsible for the initiation of AChR aggregation. In contrast, interactions of agrin with binding proteins on the muscle surface harbouring different specificities such as alpha-dystroglycan do not seem to be necessary for this process.     

Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans

The UNC-5 guidance receptor, in response to the UNC-6/netrin path cue, orients growing axons in a dorsal direction along the epidermis of Caenorhabditis elegans. When ectopically expressed in the touch neurons, which normally extend ventrally or longitudinally, UNC-5 is able to reorient their axons toward the dorsal side in an UNC-6-dependent manner. This forms the basis of a genetic screen to identify other mutations that, like unc-6 mutations, suppress unc-5-induced growth cone guidance. These mutations may identify new components required for pioneer axon guidance by unc-5. In this paper, we describe eight genes that are required for ectopic unc-5-induced growth cone steering. Mutations in four of these identify the previously known axon guidance genes unc-6, unc-40, unc-34, and unc-44 and mutations in four others identify the novel genes unc-129, seu-1, seu-2, and seu-3. Several of these mutations cause axon guidance defects similar to those found in unc-5 mutants. We propose that some or all of these genes may function in a developmentally important unc-5 signaling pathway.     

Tyrosine kinase A, galanin and nitric oxide synthase within basal forebrain neurons in the rat

Cholinergic basal forebrain neurons appear to play a key role in cognition and attention. In rat, basal forebrain neurons express multiple proteins including the high-affinity signal transducing tyrosine kinase A receptor for nerve growth factor, the neuropeptide galanin and nitric oxide synthase, a marker for the novel neurotransmitter nitric oxide. The present study was undertaken to define the relationship between neurons expressing each of these markers within the medial septum-vertical limb of the diagonal band, horizontal limb of the diagonal band and nucleus basalis in colchicine pre-treated rats. Tyrosine kinase A-immunopositive neurons were seen throughout all subfields of the basal forebrain. In contrast, nitric oxide synthase- and galanin-immunoreactive neurons were mainly distributed within the septal-diagonal band complex. Co-localization experiments revealed that virtually all nitric oxide synthase-positive neurons (visualized by nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry) also contained tyrosine kinase A, whereas many fewer tyrosine kinase A neurons were nicotinamide adenine dinucleotide phosphate-diaphorase positive within the medial septum-vertical limb of the diagonal band. Within the horizontal limb of the diagonal band, numerous nicotinamide adenine dinucleotide phosphate-diaphorase neurons expressed tyrosine kinase A, whereas only a small number of tyrosine kinase A neurons contained nicotinamide adenine dinucleotide phosphate-diaphorase. Within the nucleus basalis very few neurons were nicotinamide adenine dinucleotide phosphate-diaphorase reactive, and a minor number contained tyrosine kinase A. Additional co-localization experiments revealed minor percentages of neurons containing nicotinamide adenine dinucleotide phosphate-diaphorase and galanin immunoreactivity within the various subfields of the basal forebrain. Within the horizontal limb of the diagonal band minor numbers of nicotinamide adenine dinucleotide phosphate-diaphorase-reactive perikarya displayed galanin. Similarly, only a few galanin-containing neurons expressed nicotinamide adenine dinucleotide phosphate-diaphorase. The existence of tyrosine kinase A, nitric oxide synthase and galanin within select neuronal subgroups of the cholinergic basal forebrain suggests that these perikarya are responsive to a complex set of chemical signals. A greater understanding of the chemical signature of the cholinergic basal forebrain neurons will provide the insight required to develop novel pharmacological approaches aimed at preventing or slowing the degenerative processes that effect these neurons in aging and pathologic disorders.     

Repeated isolation in the neonatal rat produces alterations in behavior and ventral striatal dopamine release in the juvenile after amphetamine challenge.

Rat pups were isolated from the mother and nest for 1 hr per day from Postnatal Day (PN) 2 to 9. At PN 27, rats were tested for behavioral responsiveness to 2.0 or 7.5 mg/kg amphetamine. Only isolated rats receiving the 7.5 mg/kg dose displayed increased activity scores, compared with nonisolated and nonhandled controls. Their increased activity is attributed to a slower latency to enter into stereotypy. In a second experiment, similarly treated groups were challenged by the 7.5 mg/kg dose during a session in which a microdialysis probe implanted in the ventral striatum was being perfused. The challenge drug elicited a much greater increase in dialysate dopamine in isolated vs nonisolated groups. Results are discussed with regard to dissociation between sensitized and subsensitized responses. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl

The 120 kD product of the c-Cbl oncogene is a prominent substrate of protein tyrosine kinases that lacks a known catalytic activity but possesses an array of binding sites for cytoplasmic signalling proteins. An oncogenic form of Cbl was recently identified in the 70Z/3 pre-B cell lymphoma which has a small deletion at the N-terminus of the Ring finger domain. This form of Cbl, termed 70Z-Cbl, exhibits an enhanced level of tyrosine phosphorylation compared with c-Cbl. Here we demonstrate that the expression of 70Z-Cbl induces a tenfold enhancement in the kinase activity of the EGF receptor in serum-starved and EGF-stimulated cells. In serum-starved cells this results in EGF receptor autophosphorylation and the recruitment of Grb2, Shc and Sos1 but does not induce a corresponding increase in MAP kinase activity. Furthermore the expression of 70Z-Cbl greatly enhances EGF-induced tyrosine phosphorylation of the protein tyrosine phosphatase SHP-2. We also show that the Cbl/EGF receptor complex is predominantly associated with CrkII and is distinct to the Grb2/Shc/Sos1 complex that associates with the EGF receptor. These findings therefore demonstrate a biochemical effect of an oncogenic Cbl protein and support predictions from C. elegans that Cbl functions as regulator of receptor tyrosine kinases.     

Extracellular matrix fibrils and cell contacts in the chick embryo. Possible roles in orientation of cell migration and axon extension

The migration of neural crest and sclerotome cells and the extension of ventral root axons in chick embryos at stages 16-20 were studied by light microscopy as well as scanning and transmission electron microscopy at the leg bud level of fixed specimens. Extensive cellular movements take place in association with an extracellular matrix consisting of microfibrils. The neural crest and sclerotome cells migrate into the large matrix-filled extracellular space surrounding the neural tube and notochord, apparently using microfibril microfibril bundles as substratum. The cells exhibit pseudopodia which are closely associated with the matrix fibrils. The fibrils around the notochord show a spatial arrangement indicating that the sclerotome cells are contact-guided to their subsequent positions. Mutual cell contacts, including those established by cell processes, frequently show cytoplasmic electron dense plaques at adjacent membranes. These small "plaque contacts" might be correlated to contact inhibition of locomotion between the cells and participate in the guidance of cells. The growth cones of extending axons exhibit filopodia contacting both surrounding mesenchyme cells and extracellular fibrils. The orientation of the axons might thus be affected by contacts with cell surfaces as well as with extracellular material.