A method for measuring intra-tissue swelling pressure using a needle micro-osmometer.

The intervertebral disc's ability to resist load and facilitate motion arises largely from osmotic swelling pressures that develop within the tissue. Changes in the disc's osmotic environment, diurnally and with disease, have been suggested to regulate cellular activity, yet knowledge of in vivo osmotic environments is limited. Therefore, the first objective of this study was to demonstrate proof-of-concept for a method to measure intra-tissue swelling pressure and osmolality, modeling micro-osmometer fluid flux using Darcy's law. The second objective was to compare flux-based measurements of the swelling pressure within nucleus pulposus (NP) tissue against ionic swelling pressures predicted by Gibbs-Donnan theory. Pressures (0.03- 0.57 MPa) were applied to NP tissue (n = 25) using equilibrium dialysis, and intra-tissue swelling pressures were measured using flux. Ionic swelling pressures were determined from inductively coupled plasma optical emission spectrometry measurements of intra-tissue sodium using Gibbs-Donnan calculations of fixed charge density and intra-tissue chloride. Concordance of 0.93 was observed between applied pressures and flux- based measurements of swelling pressure. Equilibrium bounds for effective tissue osmolalities engendered by a simulated diurnal loading cycle (0.2-0.6 MPa) were 376 and 522 mOsm/kg H2O. Significant differences between flux and Gibbs-Donnan measures of swelling pressure indicated that total tissue water normalization and non-ionic contributions to swelling pressure were significant, which suggested that standard constitutive models may underestimate intra-tissue swelling pressure. Overall, this micro-osmometer technique may facilitate future validations for constitutive models and measurements of variation in the diurnal osmotic cycle, which may inform studies to identify diurnal- and disease-associated changes in mechanotransduction.

Increased angiogenesis and VEGF expression correlates with disease severity in prurigo patients.

BACKGROUND: Vascular endothelial growth factor-A (VEGF-A) is known as the major skin angiogenesis factor and can be produced by various resident skin cells including keratinocytes. OBJECTIVES: To identify and characterize the role of VEGF-A in the pathogenesis of prurigo. METHODS: Expression of VEGF, VEGFR2, CD-31, and D2-40 was analyzed in the skin of eleven prurigo patients and seven healthy controls by immunohistochemistry. RESULTS: VEGF immunoreactivity (IR) was markedly increased in the epidermis, dermis and subcutis of prurigo patients, whereas expression of the main receptor for VEGF-A in the skin, VEGFR2, was comparable to that of healthy controls. The increased VEGF expression in the skin was associated with a marked increase in the number (12.8 ± 2.1 vs 5.6 ± 0.5, P < 0.05) but not in the size of blood vessels, as assessed by staining of the endothelial cell marker CD31. This increase in small blood vessels correlated closely with increases in the epidermal thickness in prurigo lesions. The number of lymphatic vessels as assessed by D2-40 staining was found to be similar in prurigo patients and healthy controls. CONCLUSIONS: Based on these findings, we speculate that the observed profound vascular remodelling in prurigo might contribute to the pathogenesis of prurigo and the corresponding clinical symptoms and that targeting of VEGF may present a novel therapeutic strategy in the treatment of prurigo patients.

Exchange biasing single molecule magnets: coupling of TbPc2 to antiferromagnetic layers.

We investigate the possibility to induce exchange bias between single molecule magnets (SMM) and metallic or oxide antiferromagnetic substrates. Element-resolved X-ray magnetic circular dichroism measurements reveal, respectively, the presence and absence of unidirectional exchange anisotropy for TbPc(2) SMM deposited on antiferromagnetic Mn and CoO layers. TbPc(2) deposited on Mn thin films present magnetic hysteresis and a negative horizontal shift of the Tb magnetization loop after field cooling, consistent with the observation of pinned spins in the Mn layer coupled parallel to the Tb magnetic moment. Conversely, molecules deposited on CoO substrates present paramagnetic magnetization loops with no indication of exchange bias. These experiments demonstrate the ability of SMM to polarize the pinned uncompensated spins of an antiferromagnet during field-cooling and realize metal-organic exchange-biased heterostructures using antiferromagnetic pinning layers.

Analyses of sex and individual differences in vocalizations of Australasian gannets using a dynamic time warping algorithm.

The study of the evolution of sexual differences in behavioral and morphological displays requires analyses of the extent of sexual dimorphism across various sensory modalities. In the seabird family Sulidae, boobies show dramatic sexual dimorphism in their vocalizations, and gannet calls have also been suggested to be dimorphic to human observers. This study aimed to evaluate the presence of sexually dimorphic calls in the Australasian gannet (Morus serrator) through the first comprehensive description of its vocalizations recorded at two localities; Cape Kidnappers, where individuals were banded and sexed from DNA samples, and at the Muriwai gannetry, both on the North Island of New Zealand. Calls were first inspected using basic bioacoustic features to establish a library of call element types for general reference. Extensive multivariate tests, based on a dynamic time warping algorithm, subsequently revealed that no sexual differences could be detected in Australasian gannet calls. The analyses, however, indicated extensive and consistent vocal variation between individuals, particularly so in female gannets, which may serve to signal individual identity to conspecifics. This study generates predictions to identify whether differences in Australasian gannet vocalizations play perceptual and functional roles in the breeding and social biology of this long-lived biparental seabird species.

Coupling single molecule magnets to ferromagnetic substrates.

We investigate the interaction of TbPc(2) single molecule magnets (SMMs) with ferromagnetic Ni substrates. Using element-resolved x-ray magnetic circular dichroism, we show that TbPc(2) couples antiferromagnetically to Ni films through ligand-mediated superexchange. This coupling is strongly anisotropic and can be manipulated by doping the interface with electron acceptor or donor atoms. We observe that the relative orientation of the substrate and molecule anisotropy axes critically affects the SMM magnetic behavior. TbPc(2) complexes deposited on perpendicularly magnetized Ni films exhibit enhanced magnetic remanence compared to SMMs in the bulk. Contrary to paramagnetic molecules pinned to a ferromagnetic support layer, we find that TbPc(2) can be magnetized parallel or antiparallel to the substrate, opening the possibility to exploit SMMs in spin valve devices.

Orbital specific chirality and homochiral self-assembly of achiral molecules induced by charge transfer and spontaneous symmetry breaking.

We study the electronic mechanisms underlying the induction and propagation of chirality in achiral molecules deposited on surfaces. Combined scanning tunneling microscopy and ab initio electronic structure calculations of Cu-phthalocyanines adsorbed on Ag(100) reveal the formation of chiral molecular orbitals in structurally undistorted molecules. This effect shows that chirality can be manifest exclusively at the electronic level due to asymmetric charge transfer between molecules and substrate. Single molecule chirality correlates with attractive van der Waals interactions, leading to the propagation of chirality at the supramolecular level. Ostwald ripening provides an efficient pathway for complete symmetry breaking and self-assembly of homochiral supramolecular layers.

Correlated electrons step by step: itinerant-to-localized transition of fe impurities in free-electron metal hosts.

High-resolution photoemission spectroscopy and ab initio calculations have been employed to analyze the onset and progression of d-sp hybridization in Fe impurities deposited on alkali metal films. The interplay between delocalization, mediated by the free-electron environment, and Coulomb interaction among d electrons gives rise to complex electronic configurations. The multiplet structure of a single Fe atom evolves and gradually dissolves into a quasiparticle peak near the Fermi level with increasing host electron density. The effective multiorbital impurity problem within the exact diagonalization scheme describes the whole range of hybridizations.

Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration.

BACKGROUND: In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues-intrinsic to the somites-that are responsible for this segmental migration of neural crest cells. RESULTS: We demonstrate that Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoided migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocked this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration was disrupted by addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. CONCLUSIONS: Both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system.

Involvement of two novel chaperones in the assembly of mitochondrial NADH:Ubiquinone oxidoreductase (complex I).

The respiratory complex I of mitochondria consists of some 40 different subunits which form an L-shaped structure. Perpendicular to a hydrophobic arm embedded in the inner mitochondrial membrane a peripheral arm protrudes into the matrix. Assembly of the complex as studied in the fungus Neurospora crassa involves the formation of discrete intermediates. The matrix arm and the membrane arm are formed independently of each other and are joined in the course of assembly. The membrane arm itself is formed by association of two assembly intermediates, a smaller of 200 kDa and a larger of 350 kDa. The latter is associated with two extra proteins of 84 and 30 kDa which are not constituent parts of mature complex I. Their primary structures show no similarity to known proteins. Mutants generated by disrupting the genes of either of the two proteins accumulate the matrix arm of complex I and the small membrane arm assembly intermediate, but are incapable of forming the large intermediate. In the wild-type, the extra proteins exclusively associate with the large membrane arm assembly intermediate. Pulse-chase labelling experiments showed that the two proteins are repeatedly involved in many assembly cycles of the intermediate. These results indicate that the two proteins are novel chaperones specific for complex I membrane arm assembly.

Segmental organization of neural crest migration.

Avian neural crest cells migrate on precise pathways to their target areas where they form a wide variety of cellular derivatives, including neurons, glia, pigment cells and skeletal components. In one portion of their pathway, trunk neural crest cells navigate in the somitic mesoderm in a segmental fashion, invading the rostral, while avoiding the caudal, half-sclerotome. This pattern of cell migration, imposed by the somitic mesoderm, contributes to the metameric organization of the peripheral nervous system, including the sensory and sympathetic ganglia. At hindbrain levels, neural crest cells also travel from the neural tube in a segmental manner via three migratory streams of cells that lie adjacent to even-numbered rhombomeres. In this case, the adjacent mesoderm does not possess an obvious segmental organization, compared to the somitic mesoderm at trunk levels. Thus, the mechanisms by which the embryo controls segmentally-organized cell migrations have been a fascinating topic over the past several years. Here, I discuss findings from classical and recent studies that have delineated several of the tissue, cellular and molecular elements that contribute to the segmental organization of neural crest migration, primarily in the avian embryo. One common theme is that neural crest cells are prohibited from entering particular territories in the embryo due to the expression of inhibitory factors. However, permissive, migration-promoting factors may also play a key role in coordinating neural crest migration.

Inhibitory interactions in the patterning of trunk neural crest migration.

In avian embryos, trunk neural crest migrate segmentally through the somites, entering the rostral but not caudal somitic sclerotome. Inhibitory molecules in the caudal somite could prohibit entry of neural crest caudally, and thus restrict cell migration to rostral somite territories. Two sets of cues, peanut agglutinin (PNA) binding glycoproteins and members of the Eph family of receptor tyrosine kinases (RTKs) and their ligands appear to play this role in segmenting cell migration. Addition of exogenous PNA to neural crest migrating in trunk explants disrupts the normal segmental pattern of migration; neural crest travel in rostral and caudal regions of the somites. Members of the Eph family display the correct spatiotemporal localization to influence neural crest migration; the RTK EphB3 is expressed by neural crest, whereas the transmembrane ligand ephrin-B1 is expressed by caudal sclerotomal cells. Exogenous ephrin-B1 added to neural crest migrating in trunk explants also specifically disrupts the segmental organization of neural crest migration. Isolated neural crest avoid lanes containing ephrin-B1 in vitro; this avoidance is abolished by addition of soluble ligand. Time-lapse imaging reveals that neural crest exhibit a typical collapse response followed by process retraction upon encountering ligand. The results of these studies implicate PNA-binding glycoproteins and Eph family members in sculpting the migratory patterns of neural precursors in the peripheral nervous system.

Motor axon pathfinding in the peripheral nervous system.

Functional motor performance is dependent upon the correct assemblage of neural circuitry, a process initiated during embryonic development. How is the complicated neural circuitry that underlies functional behavior formed? During early stages of development, motor neurons extend their axons in a precise manner to their target destinations where they form fine synaptic connections. This process is not random but rather, highly stereotyped and specific. Results of recent studies indicate that positive and negative molecules influence particular steps in the navigation of motor axons to their targets. These molecules include, but are not limited to, members of the Semaphorin family and their receptors, Neuropilins and Plexins, Slits and their Robo receptors, members of the Eph family, extracellular matrix molecules, Hepatocyte Growth Factor/Scatter Factor, peanut agglutinin-binding glycoproteins, and neural cell adhesion molecule. The developing avian peripheral nervous system has served as an excellent model system for many years for studies of the basic cellular interactions that underlie motor axon pathfinding. The principal advantage for the experimental use of the avian embryo is the ease of access to early developmental events. Fine microsurgical manipulations, difficult at best in mouse embryonic development, are readily accomplished in avian embryos and have provided a powerful approach to unraveling the cellular interactions that govern motor axon pathfinding. These approaches, combined in recent years with molecular biology, have begun to produce critical insights into the mechanisms that sculpt cellular architecture during neural development.

Ephrin-as cooperate with EphA4 to promote trunk neural crest migration.

Trunk neural crest cells delaminate from the dorsal neural tube and migrate on two distinct pathways: a dorsolateral route, between the ectoderm and somites,and a ventromedial route, through the somitic mesoderm. Neural crest cells that migrate ventromedially travel in a segmental manner through rostral half-somites, avoiding caudal halves. Recent studies demonstrate that various molecular cues guide the migration of neural crest cells, primarily by serving as inhibitors to premature pathway entry orby preventing neural crest from entering inappropriate territories. Trajectories of migrating trunk neural crest are well organized and generally linear in nature, suggesting that positive, migration-promoting factors may be responsible for this organized cell behavior. However, the identity of these factors and their function are not well understood. Here we examine the expression of members of the EphA subclass of receptor tyrosine kinases and ephrins using RT-PCR and immunocytochemistry. Neural crest cells express ephrins and EphA4 at distinct stages during their migration. In functional analyses, addition of ephrin-A2-, ephrin-A5-, and EphA4-Fc disrupted the segmental organization of trunk neural crest migration in explants: neural crest cells entered rostral and caudal halves of somites. Finally, to test the specific effects of these factors on cell behavior, neural crest cells were exposed in vitro to substrate-bound EphA and ephrin-As. Surprisingly, neural crest cells avoided ephrin-A2 or ephrin-A5 substrates; this avoidance was abolished by the addition of EphA4. Together, these data suggest that ephrin-As and EphA4 cooperate to positively promote the migration of neural crest cells through rostral half somites in vivo.

Agenesis of the bladder and urethra: a case report.

Agenesis of the bladder is an extremely rare congenital anomaly. We describe a girl with complete agenesis of the bladder and urethra, who is alive 2 years after bilateral cutaneous ureterostomy.

Dorsal hindbrain ablation results in rerouting of neural crest migration and changes in gene expression, but normal hyoid development.

Our previous studies have shown that hindbrain neural tube cells can regulate to form neural crest cells for a limited time after neural fold removal (Scherson, T., Serbedzija, G., Fraser, S. E. and Bronner-Fraser, M. (1993). Development 188, 1049-1061; Sechrist, J., Nieto, M. A., Zamanian, R. T. and Bronner-Fraser, M. (1995). Development 121, 4103-4115). In the present study, we ablated the dorsal hindbrain at later stages to examine possible alterations in migratory behavior and/or gene expression in neural crest populations rostral and caudal to the operated region. The results were compared with those obtained by misdirecting neural crest cells via rhombomere rotation. Following surgical ablation of dorsal r5 and r6 prior to the 10 somite stage, r4 neural crest cells migrate along normal pathways toward the second branchial arch. Similarly, r7 neural crest cells migrate primarily to the fourth branchial arch. When analogous ablations are performed at the 10-12 somite stage, however, a marked increase in the numbers of DiI/Hoxa-3-positive cells from r7 are observed within the third branchial arch. In addition, some DiI-labeled r4 cells migrate into the depleted hindbrain region and the third branchial arch. During their migration, a subset of these r4 cells up-regulate Hoxa-3, a transcript they do not normally express. Krox20 transcript levels were augmented after ablation in a population of neural crest cells migrating from r4, caudal r3 and rostral r3. Long-term survivors of bilateral ablations possess normal neural crest-derived cartilage of the hyoid complex, suggesting that misrouted r4 and r7 cells contribute to cranial derivatives appropriate for their new location. In contrast, misdirecting of the neural crest by rostrocaudal rotation of r4 through r6 results in a reduction of Hoxa-3 expression in the third branchial arch and corresponding deficits in third arch-derived structures of the hyoid apparatus. These results demonstrate that neural crest/tube progenitors in the hindbrain can compensate by altering migratory trajectories and patterns of gene expression when the adjacent neural crest is removed, but fail to compensate appropriately when the existing neural crest is misrouted by neural tube rotation.

Segmental migration of trunk neural crest: time-lapse analysis reveals a role for PNA-binding molecules.

Trunk neural crest cells migrate through the somites in a striking segmental fashion, entering the rostral but not caudal sclerotome, via cues intrinsic to the somites. Attempts to define the molecular bases of these cues have been hampered by the lack of an accessible assay system. To examine trunk neural crest migration over time and to perturb candidate guiding molecules, we have developed a novel explant preparation. Here, we demonstrate that trunk regions of the chicken embryo, placed in explant culture, continue to develop apparently normally for 2 days. Neural crest cells, recognized by prelabeling with DiI or by poststaining with the HNK-1 antibody, migrate in the somites of the explants in their typical segmental pattern. Furthermore, this paradigm allows us to follow trunk neural crest migration in situ for the first time using low-light-level videomicroscopy. The trajectories of individual neural crest cells were often complex, with cells migrating in an episodic mode encompassing forward, backward and lateral movements. Frequently, neural crest cells migrated in close-knit groups of 2-4 cells, moving at mean rates of migration of 10-14 microns/hour. Treatment of the explants with the lectin peanut agglutinin (PNA) both slowed the rate and altered the pattern of neural crest migration. Neural crest cells entered both the rostral and caudal halves of the sclerotome with mean rates of migration ranging from 6 to 13 microns/hour. These results suggest that peanut agglutinin-binding molecules are required for the segmental patterning of trunk neural crest migration. Because this approach permits neural crest migration to be both observed and perturbed, it offers the promise of more direct assays of the factors that influence neural crest development.

In vitro analyses of neurite outgrowth indicate a potential role for tenascin-like molecules in the development of insect olfactory glomeruli.

Tenascin-like material is associated with glial cells that form borders around developing glomerular units in the olfactory (antennal) lobe of the moth Manduca sexta and is present at critical stages of glomerulus formation (Krull et al., 1994, J. Neurobiol. 25:515-534). Tenascin-like immunoreactivity declines in the mature lobe, coincident with a wave of synapse formation within the glomeruli and glomerulus stabilization. Tenascin-like molecules associated with neuropilar glia are in the correct position to influence the branching patterns of growing neurites by constraining them to glomeruli. In this study, we examine the growth of cultured moth antennal-lobe neurons in response to mouse CNS tenascin. Uniform tenascin provides a poor substrate for cell-body attachment and neurite outgrowth. Neuronal cell bodies provided with a striped substratum consisting of tenascin and concanavalin-A (con-A)/laminin attach preferentially to con-A/laminin lanes. Most neurons restrict their branching to con-A/laminin lanes both at early and later times in culture but others send processes across multiple tenascin and con-/laminin lanes in an apparently indiscriminate manner. Tenascin can inhibit the neuritic outgrowth of most antennal-lobe neurons, and this raises the possibility that the tenascin-like molecules associated with neuropilar glia in vivo act to constrain growing neurites to glomeruli. Thus, glial cells, acting in concert with olfactory axons, might act to promote glomerular patterns of branching by antennal-lobe neurons.

Spatiotemporal pattern of expression of tenascin-like molecules in a developing insect olfactory system.

During the development of the olfactory (antennal) lobe of the moth Manduca sexta, olfactory sensory axons induce glomerular branching patterns in their target neurons. Glial cells, by surrounding the developing glomerular template, are thought to mediate the developmental influence of olfactory axons on these branching patterns. Previous studies have demonstrated that, in the absence of glia, neurons in the antennal lobe branch in an aglomerular fashion, even in the presence of competent antennal axons (Oland and Tolbert, 1988, J. Comp. Neurol. 278:377-387; Oland et al., 1988, J. Neurosci. 8:353-367). We have begun to explore the molecular basis by which glial cells could influence patterns of neurite branching. For this work, we have utilized immunocytochemical techniques and a partial biochemical analysis to demonstrate that molecules antigenically similar and comparable in size to mammalian tenascin are localized on the neuropil-associated glial cells that form borders around glomeruli in the developing antennal lobe. These tenascin-like molecules associated with neuropilar glia are present at critical stages of glomerulus development; tenascin-like immunoreactivity declines after glomeruli form and become stabilized. Neither the arrival nor the absence of antennal axons in the lobe induces changes in either the molecular forms or the amounts of tenascin-like molecules. The spatiotemporal pattern of expression of tenascin-like molecules suggests that they are in a position to participate in the formation of a glomerular neuropil and could form a molecular barrier that constrains neurite outgrowth strictly to glomeruli.