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Years of important research has revealed that cells heavily invest in regulating their size. Nevertheless, it has remained unclear why accurate size control is so important. Our recent study using hematopoietic stem cells (HSCs) in vivo indicates that cellular enlargement is causally associated with aging. Here, we present an overview of these findings and their implications. Furthermore, we performed a broad literature analysis to evaluate the potential of cellular enlargement as a new aging hallmark and to examine its connection to previously described aging hallmarks. Finally, we highlight interesting work presenting a correlation between cell size and age-related diseases. Taken together, we found mounting evidence linking cellular enlargement to aging and age-related diseases. Therefore, we encourage researchers from seemingly unrelated areas to take a fresh look at their data from the perspective of cell size.

Years of important research has revealed that cells heavily invest in regulating their size. Nevertheless, it has remained unclear why accurate size control is so important. Our recent study using hematopoietic stem cells (HSCs) in vivo indicates that cellular enlargement is causally associated with aging. Here, we present an overview of these findings and their implications. Furthermore, we performed a broad literature analysis to evaluate the potential of cellular enlargement as a new aging hallmark and to examine its connection to previously described aging hallmarks. Finally, we highlight interesting work presenting a correlation between cell size and age-related diseases. Taken together, we found mounting evidence linking cellular enlargement to aging and age-related diseases. Therefore, we encourage researchers from seemingly unrelated areas to take a fresh look at their data from the perspective of cell size., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Davies, van den Handel, Bharadwaj and Lengefeld.)

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.

Purpose: Trabecular meshwork (TM) cell shape, volume, contractility and their interactions with extracellular matrix determine outflow facility. Because cell volume seems essential to TM function, this study was conducted to investigate further the ionic channels and receptors involved in regulatory volume decrease and their roles in modulating outflow facility., Methods: Primary cultures of bovine TM cells were used. K(+) and Cl(-) currents were studied with whole-cell patch clamping. Swelling was induced by hypotonic shock. [Ca(2+)](i) was measured in TM cells loaded with fura-2. Bovine anterior segments were perfused at constant pressure to measure outflow facility., Results: Hypotonic media activated both the high-conductance Ca(2+)-activated K(+) channel (BK(Ca)) and swelling-activated Cl(-) channel (Cl(swell)) currents and induced release of adenosine 5'-triphosphate (ATP) from TM cells. ATP activated P2Y(2) receptors with the following profile: ATP = uridine 5'-triphosphate (UTP) > adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma S) > adenosine 5'-diphosphate (ADP) = uridine 5'-diphosphate (UDP), and increased BK(Ca) current. Hypotonic medium initially decreased outflow facility in perfused anterior segments, which recovered with time to baseline levels. Addition of tamoxifen or iberiotoxin (Cl(swell) and BK(Ca) blockers, respectively) lengthened the recovery phase, which implies that these channels participate in cell volume regulation. In contrast, an activator of BK(Ca)s (NS1619) produced the opposite effect., Conclusions: Cell swelling activates a regulatory volume decrease mechanism that implies activation of K(+) and Cl(-) currents and participation of P2Y(2) receptors. Because previous studies have shown that intracellular volume of TM cells is an important determinant of outflow facility, it seems feasible that cell volume regulation would be part of the homeostatic mechanisms of the TM, to regulate the outflow pathway.

Glutamate is thought to participate in a variety of retinal degenerative disorders. However, when exposed to glutamate at concentrations up to 1 mM, ex vivo rat retinas typically exhibit Müller cell swelling, but not excitotoxic neuronal damage. This Müller cell swelling is reversible following glutamate washout, indicating that the glial edema is not required for glutamate-induced neuronal injury. It is unclear whether glutamate directly induces the Müller cell swelling or whether a metabolite of glutamate such as glutamine acts as an osmolyte to generate the cellular edema. To examine this issue, ex vivo rat retinas were exposed to 1 mM glutamate or 1 mM glutamine and were evaluated histologically. Glutamate was also combined with 1 mM ammonia or with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, the enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia. Glutamate-mediated Müller cell swelling was blocked by co-administration of ammonia and the reversibility of Müller cell swelling was inhibited by MSO administered following glutamate exposure. Glutamine alone failed to induce Müller cell swelling. These results indicate that glutamate-mediated Müller cell swelling is unlikely to result from glutamine accumulation. Rather, conversion of glutamate to glutamine in a reaction involving ammonia helps reverse Müller cell swelling following exposure to exogenous glutamate.

The scavenger receptor expressed by endothelial cells (SREC) was isolated from a human endothelial cell line and consists of two isoforms named SREC-I and -II. Both isoforms have no significant homology to other types of scavenger receptors. They contain 10 repeats of epidermal growth factor-like cysteine-rich motifs in the extracellular domains and have unusually long C-terminal cytoplasmic domains with Ser/Pro-rich regions. The extracellular domain of SREC-I binds modified low density lipoprotein and mediates a homophilic SREC-I/SREC-I or heterophilic SREC-I/SREC-II trans-interaction. However, the significance of large Ser/Pro-rich cytoplasmic domains of SRECs is not clear. Here, we found that when SREC-I was overexpressed in murine fibroblastic L cells, neurite-like outgrowth was induced, indicating that the receptor can lead to changes in cell morphology. The SREC-I-mediated morphological change required the cytoplasmic domain of the protein, and we identified advillin, a member of the gelsolin/villin family of actin regulatory proteins, as a protein binding to this domain. Reduction of advillin expression in L cells by RNAi led to the absence of the described SREC-I-induced morphological changes, indicating that advillin is a prerequisite for the change. Finally, we demonstrated that SREC-I and advillin were co-expressed and interacted with each other in dorsal root ganglion neurons during embryonic development and that overexpression of both SREC-I and advillin in cultured Neuro-2a cells induced long process formation. These results suggest that the interaction of SREC-I and advillin are involved in the development of dorsal root ganglion neurons by inducing the described morphological changes.

Growth differentiation factor 3 (GDF-3) is structurally a bone morphogenetic protein/growth differentiation factor subfamily member of the TGF-beta superfamily. GDF-3 exhibits highest level of expression in white fat tissue in mice and is greatly induced by high fat diet if fat metabolic pathway is blocked. To identify its biological function, GDF-3 was overexpressed in mice by adenovirus mediated gene transfer. Mice transduced with GDF-3 displayed profound weight gain when fed with high fat diet. The phenotypes included greatly expanded adipose tissue mass, increased body adiposity, highly hypertrophic adipocytes, hepatic steatosis, and elevated plasma leptin. GDF-3 stimulated peroxisome proliferator activated receptor expression in adipocytes, a master nuclear receptor that controls adipogenesis. However, GDF-3 was not involved in blood glucose homeostasis or insulin resistance, a condition associated with obesity. In contrast, similar phenotypes were not observed in GDF-3 mice fed with normal chow, indicating that GDF-3 is only active under high lipid load. Thus, GDF-3 is a new non-diabetic adipogenic factor tightly coupled with fat metabolism.

The study of the dielectric properties of micrometer- or nanometer-scale particles is of particular interest in present-day applications of biomedical engineering. Electrokinetics utilises electrically energised microelectrode structures within microfluidic chambers to noninvasively probe the physiological structure of live cancer cells. A system is described that combines the three complementary techniques of dielectrophoresis (DEP), travelling wave dielectrophoresis (TWD) and electrorotation (ROT) for the first time on a single, integrated chip (3 x 6 mm). The chip employs planar microelectrode arrays fabricated on a silicon substrate to facilitate the synthesis of the various nonuniform electric fields required for the controlled manipulation, measurement and characterization of mammalian cells. A study of the dielectric properties of human malignant cells (Daudi and NCI-H929) was performed to demonstrate the potential and the versatility of the system in providing a fully programmable microsystem.

The importance of neural impulse activity in regulating neuronal plasticity is widely appreciated; increasingly, it is becoming apparent that activity-dependent communication between neurons and glia is critical in regulating many aspects of nervous system development and plasticity. This communication takes place not only at the synapse, but also between premyelinating axons and glia, which form myelin in the PNS and CNS. Recent work indicates that neural impulse activity releases ATP and adenosine from non-synaptic regions of neurons, which activates purinergic receptors on myelinating glia. Acting through this receptor system, neural impulse activity can regulate gene expression, mitosis, differentiation, and myelination of Schwann cells (SCs) and oligodendrocytes, helping coordinate nervous system development with functional activity in the perinatal period. ATP and adenosine have opposite effects on differentiation of Schwann cells and oligodendrocytes, providing a possible explanation for the opposite effects of impulse activity reported on myelination in the CNS and PNS., (Copyright 2003 Elsevier Ltd.)

Objective: To retrospectively review, based on cytologic and histopathologic findings, the diagnoses of 13 patients with uterine sarcoma and 1 with vaginal sarcoma., Study Design: There were 8 cases of uterine carcinosarcoma (CS), 2 of leiomyosarcoma, 2 of endometrial stromal sarcoma (ESS), 1 of endocervical stromal sarcoma (ECSS) and 1 of malignant fibrous histiocytoma (MFH) of the vagina. The presence of sarcomatous components was retrospectively investigated by microscopic observation of preoperative specimens from the endocervical canal and endometrial cells. Characteristic features of sarcomatous cells were then investigated by cytodiagnostic micrometry of malignant cells., Results: Of the 14 patients, 1 with low grade ESS and 1 with homologous CS were diagnosed as negative for sarcomatous components. One case of high grade ESS had been overlooked, as were 4 cases of CS. Thus, 7 cases (50%) were diagnosed as positive for sarcomatous cells by preoperative cytologic observation. Based on these findings, 12 of the 14 cases (85.7%) were positive for sarcomatous elements on retrospective reexamination of the specimens., Conclusion: Careful attention should be paid to small sarcomatous cells since cases of ESS or ECSS with such cells show morphologic characteristics similar to those of stromatous cells. Furthermore, careful microscopic observation of an entire specimen is required to avoid misdiagnosis as carcinoma since it is easy to overlook sarcomatous elements in smears with carcinosarcoma if there are only a few sarcomatous cells.

Over the last 30 years there has been considerable interest in the use of functional electrical stimulation (FES) to restore movement to the limbs of paralyzed patients. Spinal cord injury causes a rapid loss in both muscle mass and contractile force. The atrophy is especially severe when the injury involves lower motoneurons because many months after spinal cord injury, atrophy is complicated by fibrosis and fat substitution. In this study we describe the effects of long-term lower motoneuron denervation of human muscle and present the structural results of muscle trained using FES. By means of an antibody for embryonic myosin, we demonstrate that many regenerative events continue to spontaneously occur in human long-term denervated and degenerated muscle (DDM). In addition, using electron microscopy, we describe i) the overall structure of fibers and myofibrils in long-term denervated and degenerated muscle, including the effects of FES, and ii) the structure and localization of calcium release units, or triads; the structures reputed to activate muscle contraction during excitation-contraction coupling (ECC). Both apparatus undergo disarrangement and re-organization following long-term denervation and FES, respectively. The poor excitability of human long-term DDM fibers, which extends to the first periods of FES training, may be explained in terms of the spatial disorder of the ECC apparatus. Its disorganization and re-organization following long-term denervation and FES, respectively, may play a key role in the parallel disarrangement and re-organization of the myofibrils that characterize denervation and FES training. The present structural studies demonstrate that the protocol used during FES training is effective in reverting long-term denervation atrophy and dystrophy. The mean fiber diameter in FES biopsies is 42.2 +/- 14.8 SD (p < 0.0001 vs DDM 14.9 +/- 6.0 SD); the mean percentile of myofiber area of the biopsy is 94.3 +/- 5.7 SD (p < 0.0001 vs DDM 25.7 +/- 23.7 SD); the mean percentile fat area is 2.1 +/- 2.4 SD (p < 0.001 vs DDM 12.8 +/- 12.1 SD); and the mean percentile connective tissue area is 3.6 +/- 4.6 SD (p < 0.001 vs DDM 61.6 +/- 20.1 SD). In DDM biopsies more than 50% of myofibers have diameter smaller than 10 microm, while the FES-trained subjects have more that 50% of myofibers larger than 30 microm. The recovery of muscle mass seems to be the result of both a size increase of the surviving fibers and the regeneration of new myofibers.

The distribution of NADPH diaphorase (NADPH-d)/nitric oxide synthase (NOS) neurons was evaluated during the postnatal development of the primary somatosensory cortex (SI) of the rat. Both cell counts and area measurements of barrel fields were carried out throughout cortical maturation. In addition, NADPH-d and cytochrome oxidase (CO) activities were also compared in both coronal and tangential sections of rat SI between postnatal days (P) 10 and 90. Throughout this period, the neuropil distributions of both enzymes presented a remarkable similarity and have not changed noticeably. Their distribution pattern show the PMBSF as a two-compartmented structure, displaying a highly reactive region (barrel hollows) flanked by less reactive regions (barrel septa). The number of NADPH-d neurons increased significantly in the barrel fields between P10 and P23, with peak at P23. The dendritic arborization of NADPH-d neurons became more elaborated during barrel development. In all ages evaluated, the number of NADPH-d cells was always higher in septa than in the barrel hollows. Both high neuropil reactivity and differential distribution of NADPH-d neurons during SI development suggest a role for nitric oxide throughout barrel field maturation.

The mean location of Renshaw synapses on spinal motoneurons and their synaptic conductance were estimated from changes in impedance magnitude produced by sustained recurrent inhibition. Motoneuron impedance was determined by injecting quasi-white noise current into lumbosacral motoneurons of pentobarbital-anesthetized cats. Synaptic location and conductance were estimated by comparing observed impedance changes to simulation results obtained using standard motoneuron models and compartmental models fit to each impedance function. Estimated synaptic locations ranged from 0.10 to 0.41lambda, with a mean of 0.19 or 0.24lambda, depending on the estimation method. Average dendritic path length was 262 microm. Average synaptic conductance was 23 to 27 nS (range: 6.7 to 57.9 nS), corresponding to conductance changes of 78 to 88% of resting membrane conductance. Estimated accuracy was supported by consistency using different estimation methods, agreement with Fyffe's 1991 morphological data, and comparisons of observed and simulated recurrent IPSP amplitudes. Synaptic location, but not synaptic conductance, was correlated with rheobase, a measure of motoneuron excitability. Synaptic conductance did not depend on synaptic location. A regression analysis demonstrated that synaptic conductance and cell impedance were the principal factors determining recurrent IPSP amplitude. Simulations using the observed values and locations of Renshaw conductance demonstrate that recurrent inhibition can require as much as an additional 14 to 18% sustained excitatory synaptic conductance to depolarize motoneurons sufficiently to activate somatic or dendritic inward currents and recruit motoneurons or amplify excitatory synaptic currents.

Diffusion-weighted nuclear magnetic resonance (NMR) imaging (DWI) is sensitive to the random translational motion of water molecules due to Brownian motion. Although the mechanism is still not completely understood, the cellular swelling that accompanies cell membrane depolarization results in a reduction in the net displacement of diffusing water molecules and thus a concomitant reduction in the apparent diffusion coefficient (ADC) of tissue water. Cerebral regions of reduced ADC appear hyperintense in a DWI and this technique has been used extensively to study acute stroke. In addition to cerebral ischemia, reductions in the ADC of cerebral water have been observed following cortical spreading depression, ischemic depolarizations (IDs), transient ischemic attack (TIA), status epilepticus, and hypoglycemia. Although the mechanism responsible for initiating membrane depolarization varies in each case, the ensuing cell volume changes follow a similar pattern. Water ADC values are also affected by the presence and orientation of barriers to translational motion (such as cell membranes and myelin fibers) and thus NMR measures of anisotropic diffusion are sensitive to more chronic pathological states where the integrity of these structures is modified by disease. Both theoretical prediction and experimental evidence suggest that the ADC of tissue water is related to the volume fraction of the interstitial space via the electrical conductivity of the tissue. The implication is that acute neurological disorders that exhibit electrical conductivity changes should also exhibit ADC changes that are detectable by DWI. A qualitative correlation between electrical conductivity and the ADC of water has been demonstrated in a number of animal model studies and the results indicate that reduced ADC values are associated with reductions in the extracellular volume fraction and increased extracellular tortuosity. The close relationship between ADC changes and cell volume changes in various pathological states suggests that NMR measurements are also sensitive to chemical communication between cells through the extracellular space (i.e., extrasynaptic or volume transmission, VT)., (Copyright 2003 Elsevier Ltd.)

Size-sieved stem cells (SSCs) derived from human bone marrow have the ability to differentiate into bone, fat and cartilage. SSCs can differentiate into active neural cells after exposure to antioxidant agents. The aim of the present study is to understand if SSCs can be stimulated to differentiate into neurons in response to neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), pituitary adenylate cyclase-activating polypeptide (PACAP) and dibutyryl cAMP (dbcAMP). SSCs in a serum-free medium transform from a fibroblastic-like form to a multipolar morphology. Treatment of SSCs with GDNF, PACAP, and dbcAMP increased the production of neurofilament light protein (NF-L) and a cytoskeleton protein-alpha-tubulin. Examination of a vesicle protein-synapsin-1 or a neuronal progenitor marker-internexin in SSCs indicated that treatment with GDNF, PACAP, and dbcAMP further elongated cell processes and increased process branching. The findings indicate that neurotrophic signaling and cAMP-dependent signaling might promote the neuronal differentiation of SSCs.

In Brn3b(-/-) mice, where 80% of retinal ganglion cells degenerate early in development, the remaining 20% include most or all ganglion cell types. Cells of the same type cover the retinal surface evenly but tile it incompletely, indicating that a regular mosaic and normal dendritic field size can be maintained in the absence of contact among homotypic cells. In Math5(-/-) mice, where only approximately 5% of ganglion cells are formed, the dendritic arbors of at least two types among the residual ganglion cells are indistinguishable from normal in shape and size, even though throughout development they are separated by millimeters from the nearest neighboring ganglion cell of the same type. It appears that the primary phenotype of retinal ganglion cells can develop without homotypic contact; dendritic repulsion may be an end-stage mechanism that fine-tunes the dendritic arbors for more efficient coverage of the retinal surface.

Neuropathic pain results from traumatic or disease-related insults to the nervous system. Mechanisms that have been postulated to underlie peripheral neuropathy commonly implicate afferent neurons that have been damaged but still project centrally to the spinal cord, and/or intact neurons that interact with degenerating distal portions of the injured neurons. One pain state that is observed following peripheral nerve injury in the rat is thermal hyperalgesia. The noxious heat-gated ion channel TRPV1 may be responsible for this increased sensitivity, as it is up-regulated in L4 dorsal root ganglion (DRG) neurons following L5 spinal nerve lesion (SpNL). The TRPV1 homologue TRPV2 (or VRL-1) is another member of the TRPV subfamily of TRP ion channels. TRPV2 is a nonselective cation channel activated by high noxious temperatures (>52 degrees C) and is present in a subset of medium- to large-diameter DRG neurons. To establish whether TRPV2 is endogenous to the spinal cord, we examined its expression in the dorsal horn following rhizotomy. We found no significant decrease in TRPV2 immunoreactivity, suggesting that TRPV2 is endogenous to the spinal cord. In order to determine whether TRPV2, like TRPV1, is regulated by peripheral axotomy, we performed L5 SpNL and characterized TRPV2 distribution in the DRG, spinal cord, brainstem, and sympathetic ganglia. Our results show that peripheral axotomy did not regulate TRPV2 in the DRG, spinal cord, or brainstem; however, TRPV2 was up-regulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated neuropathic pain.

Although great strides have recently been made in elucidating the factors initiating tumor cell migration and the relevant cellular pathways involved, the constituent components of migratory dynamics for individual tumor cell motion have still not been resolved. Utilizing a three-dimensional (3D) collagen assay and computer-assisted, continuous single cell tracking, we investigated the basic parameters for both the spontaneous and norepinephrine-induced migration of highly metastatic MBA-MB-468 breast, PC-3 prostate, and SW 480 colon carcinoma cells. We show that tumor cells do not migrate with uniform migrational structure and speed as previously thought, but rather, the induction of locomotion elicits significant increases in speed, break frequency, and total cell displacement, but decreases in break length and no change in the recruitment of nonlocomotory cells. We furthermore illustrate the corresponding morphological changes of induced tumor cell migration with emphasis on motion in a collagen matrix. These results demonstrate the complexity of tumor cell migration, and the compulsion for incorporating not only knowledge of intracellular pathways, but also fundamental parameters of migratory behavior into any expansive theory of tumor cell migration and metastasis formation. We furthermore establish the analytical methodology of investigating both the stimulation and potential pharmaceutical inhibition of tumor cell migration.

The cellular mechanisms underlying glial cell swelling, a central cause of edema formation in the brain and retina, are not yet known. Here, we show that glial cells in the postischemic rat retina, but not in control retina, swell upon hypotonic stress. Swelling of control cells could be evoked when their K(+) channels were blocked. After transient ischemia, glial cells strongly downregulated their K(+) conductance and their prominent Kir4.1 protein expression at blood vessels and the vitreous body. In contrast, the expression of the aquaporin-4 (AQP4) (water channel) protein was only slightly altered after ischemia. Activation of D(2) dopaminergic receptors prevents the hypotonic glial cell swelling. The present results elucidate the coupling of transmembraneous water fluxes to K(+) currents in glial cells and reveal the role of altered K(+) channel expression in the development of cytotoxic edema. We propose a mechanism of postischemic glial cell swelling where a downregulation of their K(+) conductance prevents the emission of intracellularly accumulated K(+) ions, resulting in osmotically driven water fluxes from the blood into the glial cells via aquaporins. Inhibition of these water fluxes may be beneficial to prevent ischemia-evoked glial cell swelling.

In the present study, the in vitro biocompatibility of calcium metaphosphate (CMP) with human bone marrow stromal cells (HBMSCs) and its effect on osteoblastic differentiation have been investigated. Powder and disk forms of CMP do not exert a cytotoxic effect on the HBMSCs undergoing osteoblastic differentiation. In addition, the HBMSCs adhere to the surface of the CMP disk as successfully as to the culture plate or hydroxyapatite (HA) disk. The HBMSCs adhered to either the HA or CMP disk display an undistinguishable actin arrangement and cellular phenotypes, indicating that the CMP does not disrupt normal cellular responses. An analysis of the differentiation of the HBMSCs cultured on culture plate, the HA and the CMP disk shows that three matrices are capable of supporting osteoblastic differentiation of the HBMSCs as accessed by alkaline phosphatase (ALP) staining. Further molecular analysis of osteoblastic differentiation of HBMSCs reveals that the CMP disk has a better ability than the HA disk to induce an expression of osteoblast-related genes, including ALP, osteoprotegerin (OPG), a decoy receptor for RANK ligand, and osteopontin (OPN), a non-collagenous bone matrix protein. The results demonstrate that, in addition to favorable biocompatibility, the CMP can stimulate osteoblastic differentiation of the HBMSCs in vitro.

The peripheral benzodiazepine receptor (PBR), a benzodiazepine but not gamma-aminobutyric acid-binding mitochondrial membrane protein, has roles in steroid production, energy metabolism, cell survival and growth. PBR expression in the nervous system has been reported in non-neuronal glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand, [(3)H]PK11195, in dorsal root ganglion (DRG) neurons following injury to the sciatic nerve. In naïve animals, PBR mRNA, protein expression and ligand binding are undetectable in the DRG. Three days after sciatic nerve transection, however, PBR mRNA begins to be expressed in injured neurons, and 4 weeks after the injury, expression and ligand binding are present in 35% of L4 DRG neurons. PBR ligand binding also appears after injury in the superficial dorsal horn of the spinal cord. The PBR expression in the DRG is restricted to small and medium-sized neurons and returns to naïve levels if the injured peripheral axons are allowed to regrow and reinnervate targets. No non-neuronal PBR expression is detected, unlike its putative endogenous ligand the diazepam binding inhibitor (DBI), which is expressed only in non-neuronal cells, including the satellite cells that surround DRG neurons. DBI expression does not change with sciatic nerve transection. PBR acting on small-calibre neurons could play a role in the adaptive survival and growth responses of these cells to injury of their axons.

Olfactory ensheathing cells (OECs) may support axonal regrowth, and thus might be a viable treatment for spinal cord injury (SCI); however, peripherally-derived OECs remain untested in most animal models of SCI. We have transplanted OECs from the lamina propria (LP) of mice expressing green fluorescent protein (GFP) in all cell types into immunosuppressed rats with cervical or lumbar dorsal root injuries. LP-OECs were deposited into either the dorsal root ganglion (DRG), intact or injured dorsal roots, or the dorsal columns via the dorsal root entry zone (DREZ). LP-OECs injected into the DRG or dorsal root migrated centripetally, and migration was more extensive in the injured root than in the intact root. These peripherally deposited OECs migrated within the PNS but did not cross the DREZ; similarly, large- or small-caliber primary afferents were not seen to regenerate across the DREZ. LP-OEC deposition into the dorsal columns via the DREZ resulted in a laminin-rich injection track: due to the pipette trajectory, this track pierced the glia limitans at the DREZ. OECs migrated centrifugally through this track, but did not traverse the DREZ; axons entered the spinal cord via this track, but were not seen to reenter CNS tissue. We found a preferential association between CGRP-positive small- to medium-diameter afferents and OEC deposits in injured dorsal roots as well as within the spinal cord. In the cord, OEC deposition resulted in increased angiogenesis and altered astrocyte alignment. These data are the first to demonstrate interactions between sensory axons and peripherally-derived OECs following dorsal root injury., (Copyright 2004 Wiley-Liss, Inc.)

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.

Biomaterials and tissue engineering are becoming increasingly important in biomedical practice, particularly as the population ages. It is clear that cellular responses to materials depend on structural properties of the material at both the micrometer- and nanometer scale, but general methods for controlling material properties on both of these scales are lacking. Using a hierarchical approach that mimics natural material formation processes, we developed a method to produce materials with controlled physical structures at both the micrometer- and nanometer scale. Our method is based upon a pre-organized micropatterned template and conformal transformation of the architecture with nanostructured minerals, namely hydroxyapatite. The newly developed materials were biocompatible with bone cells, induced a range of desirable cellular responses, and may therefore have direct application in bone tissue engineering. In addition, the design principles employed in this study can be extrapolated to the other classes of biomedical materials, including polymers, metals, ceramics or hybrid combinations.

The hypothalamus of the adult ring dove contains acoustic units that respond to species-specific coo vocalization. Loss of nest coo leads to unsuccessful breeding. However, the recovery of nest coo in some doves suggests that these units are capable of self-renewal. We have previously shown that lesioning the hypothalamus generates the addition of new neurons at the lesioned area. In this study, we sought to determine whether lesion-induced new neurons are involved in the recovery of coo-responsive units. We systematically recorded electrical activity in the ventromedial nucleus (VMN) of the hypothalamus, before and after lesion, for varying periods up to 3 months. Recordings were made when the birds were at rest (spontaneous discharge) and when the birds were exposed to acoustic stimulations (evoked discharge). Concurrently, the lesioned area was monitored for changes in cell types by using bromodeoxyuridine (BrdU) to label newly divided cells and NeuN to identify mature neurons. For 1 month after lesion, there was no sign of electrical activity, and only BrdU-labeled cells were present. When the first electrical activity occurred, it displayed abnormal spontaneous bursting patterns. The mature discharge patterns (both spontaneous and evoked) occurred after detection of BrdU+/NeuN+ double-labeled cells 2-3 months postlesion and were similar to those found in intact and sham-lesioned birds. Double-labeled cells bore morphologic characteristics of a neuron and were confirmed with z-stack analysis using confocal laser scanning microscopy. Moreover, double-labeled cells were not stained for glial fibrillary acidic protein (GFAP), suggesting that they were neurons. The number of coo-responsive units was significantly correlated with that of BrdU+/NeuN+ cells. Furthermore, the marker for recording sites revealed that coo-responsive units were colocalized with BrdU+/NeuN+ cells. Taken together, the evidence strongly suggests that lesion-induced addition of new neurons promotes the functional recovery of the adult hypothalamus., (Copyright 2004 Wiley Periodicals, Inc.)

Physical forces play a critical role in the survival and proliferation of many cell types, including fibroblasts. Gingival fibroblasts are exposed to mechanical stress during mastication, orthodontic tooth movement, and wound healing following periodontal surgery. The aim of this study was to examine the effect of mechanical strain on human gingival fibroblasts (hGF). Cells were subjected to short-term (up to 60 min) and long-term (up to 48 hrs) 20% average elongation at 0.1 Hz. We monitored survival signaling by evaluating the phosphorylation status and localization of Forkhead box (FoxO) family members, which are mediators of apoptosis. We also examined strain-induced proliferation by measuring the level of proliferating cell nuclear antigen (PCNA). We observed that cyclic strain caused the phosphorylation and retention in the cytoplasm of FoxO family members. Moreover, mechanical strain resulted in increased ERK kinase phosphorylation and PCNA expression. In conclusion, cyclic strain delivers anti-apoptotic and proliferative stimuli to hGF.

One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.

Two types of cells--human platelets and spore cells--were encapsulated in polymer shells by adsorbing polyanions and polycations in a stepwise fashion. The encapsulated cells were attached to gold and silicon surfaces and their morphological and adhesion properties were studied in air using tapping mode atomic force microscopy (AFM). The roughness of the encapsulated cells increased upon the addition of a new polymer layer. The increase in roughness can be attributed to the formation of a shell around the cells, which is stabilized by electrostatic interactions, as well as to the drying effects associated with the immobilization and dehydration of the cells. Trigger mode was used to perform the force imaging and map out the adhesion characteristics of the cells. Systematic "maps" of the adhesion properties of the encapsulated cells to clean and amine terminated AFM tips were collected. The adhesion force data for the different tips and encapsulated cells showed dependence not only on the number and thickness of the polymer layers, but also on the interactions between these layers. The encapsulated cells' morphology and roughness characteristics remained intact over a substantial storage period. This stability and adhesion properties make them suitable building blocks for the design and construction of biomimetic templates where AFM is used as the primary tool to do the fabrication.

Activation of neurons in the rostral ventromedial medulla (RVM) directly modulates spinal nociceptive transmission by projections to the spinal cord dorsal horn and indirectly by projections to neurons in the dorsolateral pons (DLP) that project to the spinal cord dorsal horn. However, it is not known whether the same neurons in the RVM produce both direct and indirect modulation of nociception. Deposits of the retrograde tracers Fluoro-Gold (FG) in the spinal cord dorsal horn and DiI in the DLP were used to determine whether the same RVM neurons project to both of these regions. Only 0.9+/-0.1% of RVM neurons retrogradely labeled with Fluoro-Gold from the spinal cord were also labeled with DiI placed in the DLP. In addition, spinally projecting RVM neurons were significantly larger than RVM neurons that project to the DLP. Finally, spinally projecting neurons were found predominantly on the midline and within the RVM; neurons that project to the DLP had a wider distribution and were present both within and outside of the RVM. Thus, separate and morphologically distinct populations of RVM neurons appear to modulate nociception by direct and indirect descending pathways.

An interactive image processing program was developed to quantify the effects of various biochemical and physical factors on cultured explants of nerve tissue. We used this method to obtain a growth curve of chick embryo dorsal root ganglia (DRG) in media containing various concentrations of nerve growth factor (NGF). In the past, neurite lengths and numbers were measured manually using collages of 35 mm color photographs or made directly under the microscope. Our new program makes it possible to quantify the growth of whole live, unstained DRG's on photograph collages or digital images with respect to center area, neurite area, total explant area, and the number and length of neurites almost exclusive of background artifacts. After comparing the old and new methods, we conclude that our analysis algorithm correlates well with previously accepted protocols for assessing stimulation and inhibition of growth. It rapidly measures several biologically-relevant properties and provides a means to obtain information on six parameters (neurite area, neurite length, neurite number, center area, total area, neurite density) using a single quantitative method. Neurite area in the presence of 10 ng/ml or 20 ng/ml NGF was the most significantly increased parameter as was expected from previous studies since it includes both neurite length and number as well as any crossing fibers., (Copyright 2004 Elsevier B.V.)

This study unravels the microscopic organization of the juxtacapsular nucleus of the bed nuclei of the stria terminalis (Ju) by using silver impregnation and electron microscopic techniques. Examination of Golgi-impregnated specimens demonstrates that the Ju has precise boundaries primarily determined by a conical condensation of fibers of the stria terminalis (StT) around the nucleus. The internal capsule, ansa peduncularis, and medial forebrain bundle together with the StT provide extrinsic afferents to the neuropil of the Ju. Two main neuron types are found in the Ju: interneurons (including basket and neurogliaform cells) and projection neurons (bipolar and small pyramidal cells). The bipolar cell type accounts for about 80% of the sampled neurons. Short-axon neurons located within the dorsal part of the Ju send descending fibers that appear to terminate on the bipolar neurons, suggesting the existence of vertically oriented functional units within the nucleus. With the electron microscope, Ju neurons are seen in clusters of two or three neurons coupled by gap junctions. The neuropil contains numerous dendrites, axons, myelinated axons, and several types of synaptic interactions, including axospinous, axoshaft, and axosomatic. Within the neuropil, Ju neurons appear to be presynaptically modulated by axoaxonal interactions. The present findings suggest a model wherein bipolar neurons represent the output system of the Ju controlled by the interneurons, which would, in turn, be modulated by collaterals arising from the tributary fiber tracts. Additional neural interaction between Ju neurons utilizes gap junction-mediated electrotonic coupling., (Copyright 2004 Wiley-Liss, Inc.)

Alterations in the basal forebrain cholinergic system have been widely studied in brain aging and Alzheimer's disease, but the magnitude of decline and relationship to cognitive impairment are still a matter of debate. The rhesus monkey (Macaca mulatta) provides a compelling model to study age-related memory decline, as the pattern of impairment closely parallels that observed in humans. Here, we used antibodies against the vesicular acetylcholine transporter and a new stereological technique to estimate total cholinergic fiber length in hippocampal subregions of behaviorally characterized young and aged rhesus monkeys. The analysis revealed an age-related decline in the length of cholinergic fibers of 22%, which was similar across the hippocampal subregions studied (dentate gyrus granule cell and molecular layers, CA2/3-hilus, and CA1), and across the rostral-caudal extent of the hippocampus. This effect, however, was unrelated to performance on the delayed nonmatching-to-sample task, a test of recognition memory sensitive to hippocampal system dysfunction and cognitive aging in monkeys. These findings indicate that a decline in cholinergic input fails to account for the influence of normal aging on memory supported by the primate hippocampal region., (Copyright 2004 Wiley-Liss, Inc.)

Differentiation of stem cells toward a neuronal lineage normally involves a gradually progressive restriction in developmental potential and is regulated by a diverse set of specific and temporally precise genetic events. However, recent studies have indicated that both rodent and human bone marrow stromal cells (MSCs) can be rapidly (within minutes to hours) induced to differentiate into neurons in vitro by relatively simple chemical means (using beta-mercaptoethanol [BME] or dimethylsulfoxide [DMSO] and butylated hydroxyanisol [BHA]; Woodbury et al. [ 2000] J. Neurosci. Res. 61:364-370). The ability to transdifferentiate an easily accessible cell source into neurons could have substantial potential for promoting neural repair. We therefore explored the potential of simple chemical methods to transdifferentiate other cell types, including primary rat fibroblasts, primary human keratinocytes, HEK293 cells, rat PC-12 cells, and as positive control rat bone marrow stromal (BMS) cells. Surprisingly, all cells except for keratinocytes adopted at least partial "neuron-like" pyramidal cell morphology with fine-cellular extensions resembling neurites upon stimulation with BME or DMSO/BHA. However, time-lapse microscopy indicated that the chemical exposure of MSCs did not result in new neurite growth but rather cellular shrinkage, with retraction of the majority of existing cell extensions, leaving only few, fine neurite-like processes. To determine whether the chemically induced transdifferentiation resulted from simple cellular toxicity, MSCs were exposed to various stressors, including detergents, high-molarity sodium chloride, and extremes of pH. In all cases, cellular shrinkage and adoption of pseudoneuronal morphology were observed. Concomitantly with cellular shrinkage, apparent increases in immunolabeling for the neuronal markers NSE and NeuN were detected in the cell soma that could not be confirmed by RT-PCR. Furthermore, blockade of protein synthesis with cycloheximide did not prevent cells from adopting "neuron-like" morphology after chemical induction. Thus, morphological changes and increases in immunolabeling for certain cellular markers upon "chemical induction" of MSCs are likely the result of cellular toxicity, cell shrinkage, and changes in the cytoskeleton and do not represent regulated steps in a complicated cellular differentiation process., (Copyright 2004 Wiley-Liss, Inc.)

Cell shrinkage is an integral part of apoptosis. However, intimate mechanisms linking apoptotic events to the alterations in cell volume homeostasis remain poorly elucidated. We investigated how overexpression of Bcl-2 oncoprotein, a key antiapoptotic regulator, in lymph node carcinoma of the prostate (LNCaP) prostate cancer epithelial cells interferes with the volume-regulated anion channel (VRAC), a major determinant of regulatory volume decrease. Bcl-2 overexpression resulted in the doubling of VRAC-carried swelling-activated Cl(-) current (I(Cl,swell)) and weakened I(Cl,swell) inhibition by store-operated Ca(2+) channel (SOC)-transported Ca(2+). This also was accompanied by substantial up-regulation of ClC-3 protein, a putative molecular candidate for the role of VRAC. ClC-3-specific antibody suppressed I(Cl,swell) in the wild-type and Bcl-2-overexpressing LNCaP cells. Epidermal growth factor treatment of wild-type LNCaP cells, promoting their proliferation, resulted in the enhancement of endogenous Bcl-2 expression and associated increases in ClC-3 levels and I(Cl,swell) magnitude. We conclude that Bcl-2-induced up-regulation of I(Cl,swell), caused by enhanced expression of ClC-3 and weaker negative control from SOC-transported Ca(2+), would strengthen the ability of the cells to handle proliferative volume increases and thereby promote their survival and diminish their proapoptotic potential.

Myosins are eukaryotic molecular motors moving along actin filaments. Only a small set of myosin classes is present in plants, in which myosins have been found to play a role in cytoplasmic streaming and chloroplast movement. Whereas most studies have been done on green algae, more recent data suggest a role of higher plant myosin at the postcytokinetic cell wall. Here we characterize a loss-of-function mutation for a myosin of plant-specific class XI and demonstrate myosin functions during plant development in Arabidopsis. T-DNA insertion in MYA2 caused pleiotropic effects, including flower sterility and dwarf growth. Elongation of epidermal cells, such as in hypocotyls and anther filaments, was reduced by up to 50% of normal length. This effect on anther filaments is responsible for flower sterility. In the meristems of root tips, it was evident that cell division was delayed and that cell plates were mislocated. Like zwichel, a kinesin-related mutation causing two-branched trichomes, the mya2 knockout causes branching defects, but here the trichomes remained unbranched. Growth was also impaired in pollen tubes and root hairs, cells that are highly dependent on vesicle transport. A failure in vesicle flow could be directly confirmed, because cytoplasmic streaming of vesicles and, more so, of large endoplasmic reticulum-based organelles was slowed. The defect in vesicle trafficking was accompanied by failures in basipetal auxin transport, measured in stem segments of inflorescences. This result strongly suggests a causal link between auxin-dependent processes and the distribution of vesicles and membrane-bound molecules by plant myosin.

Astrocyte swelling is observed in different types of brain injury. We studied a potential contribution of swelling to protein tyrosine nitration (PTN) by using cultured rat astrocytes exposed to hypoosmotic (205 mosmol/L) medium. Hypoosmolarity (2 h) increases total PTN by about 2-fold in 2 h. The hypoosmotic PTN is significantly inhibited by the NMDA receptor antagonist MK-801, the nitric oxide synthase (NOS) inhibitor L-NMMA, the extracellular Ca2+ chelator EGTA and the calmodulin antagonist W13, suggesting the involvement of NMDA receptor activation, influx of extracellular Ca2+ and Ca2+/calmodulin-dependent NO synthesis. Further, superoxide dismutase plus catalase and uric acid strongly inhibit hypoosmotic PTN, suggesting the involvement of the toxic metabolite peroxynitrite (ONOO-) as a nitrating agent. Hypoosmotic astrocyte swelling rapidly stimulates generation of reactive oxygen intermediates; this process is prevented by MK-801 and EGTA. In addition, MK-801 inhibits the hypoosmotic elevation of [Ca2+]i. The findings support the view that astrocyte swelling as induced, for example, by toxins relevant for hepatic encephalopathy is sufficient to produce oxidative stress and PTN and thus contributes to altered astroglial and neuronal function., (Copyright 2004 Wiley-Liss, Inc.)

Spinal neurones that receive inputs from primary afferent fibres and have axons projecting supraspinally to the medulla oblongata may represent a pathway through which nociceptive and non-nociceptive peripheral stimuli are able to modulate cardiorespiratory reflexes. Expression of the neurokinin-1 (NK1) receptor is believed to be an indicator of lamina I cells that receive nociceptive inputs from substance P releasing afferents, and similarly, sst2A receptor expression may be a marker for neurones receiving somatostatinergic inputs. In this study, immunoreactivity for these two receptors was investigated in rat spinal neurones retrogradely labelled by injections of cholera toxin B or Fluorogold into the nucleus of the solitary tract (NTS). In addition, nociceptive activation of these labelled cells was studied by immunodetection of Fos protein in response to cutaneous and visceral noxious chemical stimuli. NK1 and sst2A receptors in lamina I were localised to mainly separate populations of retrogradely labelled cells with fusiform, flattened and pyramidal morphologies. Examples of projection neurones expressing both receptors were, however observed. With visceral stimulation, many retrogradely labelled cells expressing c-fos were immunoreactive for the NK1 receptor, and a smaller population was sst2A positive. In contrast, with cutaneous stimulation, only NK1 positive retrogradely labelled cells showed c-fos expression. These data provide evidence that lamina I neurones receiving noxious cutaneous and visceral stimuli via NK1 receptor activation project to NTS and so may be involved in coordinating nociceptive and cardiorespiratory responses. Moreover, a subpopulation of projection neurones that respond to visceral stimuli may receive somatostatinergic inputs of peripheral, local or supraspinal origins.

The differentiation and growth of adult stem cells within engineered tissue constructs are hypothesized to be influenced by cell-biomaterial interactions. In this study, we compared the chondrogenic differentiation of human adipose-derived adult stem (hADAS) cells seeded in alginate and agarose hydrogels, and porous gelatin scaffolds (Surgifoam), as well as the functional properties of tissue engineered cartilage constructs. Chondrogenic media containing transforming growth factor beta 1 significantly increased the rates of protein and proteoglycan synthesis as well as the content of DNA, sulfated glycosaminoglycans, and hydroxyproline of engineered constructs as compared to control conditions. Furthermore, chondrogenic culture conditions resulted in 86%, and 160% increases ( p < 0.05 ) in the equilibrium compressive and shear moduli of the gelatin scaffolds, although they did not affect the mechanical properties of the hydrogels over 28 days in culture. Cells encapsulated in the hydrogels exhibited a spherical cellular morphology, while cells in the gelatin scaffolds showed a more polygonal shape; however, this difference did not appear to hinder the chondrogenic differentiation of the cells. Furthermore, the equilibrium compressive and shear moduli of the gelatin scaffolds were comparable to agarose by day 28. Our results also indicated that increases in the shear moduli were significantly associated with increases in S-GAG content ( R2 = 0.36, p < 0.05 ) and with the interaction between S-GAG and hydroxyproline ( R2 = 0.34, p < 0.05 ). The findings of this study suggest that various biomaterials support the chondrogenic differentiation of hADAS cells, and that manipulating the composition of these tissue engineered constructs may have significant effects on their mechanical properties.

By virtue of their electroneutral exchange of intracellular H+ for extracellular Na+, the Na+/H+ exchangers (NHE1-NHE8) play a pivotal role in many physiological processes. This review focuses on the ubiquitous plasma membrane isoform, NHE1. Particular attention is given to the roles and regulation of NHE1 in erythrocytes, in their own right and as model systems, but pertinent findings from non-erythroid cells are also discussed. NHE1 plays a key role in the regulation of cell volume and pH, and consequently in the control of such diverse processes as blood O2/CO2 transport, and cell proliferation, motility, and survival. Disturbances in NHE1 function are involved in important pathological states such as hypoxic cell damage and cancer development. NHE1 has a predicted topology of 12 transmembrane domains, and a hydrophilic C-terminus thought to be the major site for NHE1 regulation. NHE1 is highly conserved throughout the vertebrate phylum, particularly in the transmembrane region and the proximal part of the C-terminus. In non-erythroid, and probably also in erythroid cells, this part of the hydrophilic C-terminus interacts with multiple binding partners important for NHE1 function. Erythrocyte NHE1s from mammalian, amphibian, and teleost species are activated by cell shrinkage, decreased pH(i), inhibition of Ser/Thr protein phosphatases, and activation of Ser/Thr protein kinases, i.e., many of the stimuli activating NHE1 in non-erythroid cells. In erythrocytes of many lower vertebrates, NHE1 is activated during hypoxia and is an important modulator of hemoglobin oxygen affinity. Sensitivity of NHE1 to oxygenation status has recently been described also in non-erythroid mammalian cells., (Copyright 2004 Wiley-Liss, Inc.)

To elucidate the role of the degeneration of motor and sensory fibers in neuropathic pain, we examined the pain-related behaviors and the changes of brain-derived neurotrophic factor (BDNF) in the L4/5 dorsal root ganglion (DRG) and the spinal cord after L5 ventral rhizotomy. L5 ventral rhizotomy, producing a selective lesion of motor fibers, produced thermal hyperalgesia and increased BDNF expression in tyrosine kinase A-containing small- and medium-sized neurons in the L5 DRG and their central terminations within the spinal cord, but not in the L4 DRG. Furthermore, L5 ventral rhizotomy up-regulated nerve growth factor (NGF) protein in small to medium diameter neurons in the L5 DRG and also in ED-1-positive cells in the L5 spinal nerve, suggesting that NGF synthesized in the degenerative fibers is transported to the L5 DRG and increases BDNF synthesis. On the other hand, L5 ganglionectomy, producing a selective lesion of sensory fibers, produced heat hypersensitivity and an increase in BDNF and NGF in the L4 DRG. These data indicate that degeneration of L5 sensory fibers distal to the DRG, but not motor fibers, might influence the neighboring L4 nerve fibers and induce neurotrophin changes in the L4 DRG. We suggest that these changes of neurotrophins in the intact primary afferents of neighboring nerves may be one of many complex mechanisms, which can explain the abnormal pain behaviors after nerve injury. The ventral rhizotomy and ganglionectomy models may be useful to investigate the pathophysiological mechanisms of neuropathic pain after Wallerian degeneration in motor or sensory or mixed nerve.

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.

The pro-opiomelanocortin (POMC)-derived peptides, alpha-melanocyte-stimulating hormone, and adrenocorticotropic hormone, are important mediators of human skin pigmentation via action at the melanocortin-1 receptor. Recent data suggests that such a regulatory role also exists for the endogenous opiate, beta-endorphin (beta-END). A role for this beta-END in the regulation of follicular pigmentation, however, has not been determined. This study was designed to examine the involvement of the beta-END/mu-opiate receptor system in human follicular melanocyte biology. We employed RT-PCR, and immunohisto/cytochemistry and immunoelectron microscopy using beta-END and mu-opiate receptor specific antibodies and a functional role for beta-END was assessed by direct stimulation with the peptide. This study has demonstrated that human hair follicle melanocytes (HFM) express mRNA for the mu-opiate receptor and POMC. Furthermore, beta-END and its high affinity mu-opiate receptor are expressed at the protein level in glycoprotein100-positive follicular melanocytes and as a function of their anatomic location and differentiation status during the hair growth cycle. Functional studies revealed that beta-END is a modifier of HFM phenotype via its ability to upregulate melanogenesis, dendricity, and proliferation. These findings suggest a new regulatory role for beta-END in human HFM biology, providing a new research direction into the fundamental regulation of human hair pigmentation.

Iron-phosphate glass fibres based on the CaO-Na2O-Fe2O3-P2O5 system have been processed and characterised via thermal, XRPD, dissolution rates, diameter and biocompatibility studies. The compositions investigated were fixed at 50mol% P2O5, and the CaO content was varied between 30, 35 and 40mol%. The Fe2O3 was added in low amounts from 1-5mol%, substituting it for the Na2O mol%. The number of Tc (crystallisation temperature) peaks detected from the thermal analysis traces only showed correlation with XRPD analysis, for five out of the 15 compositions investigated. It has been suggested that either the crystalline phases had very similar Tc temperatures or that the other phase(s) were present in very small quantities. There was a good match seen with number of Tm (melting temperature) peaks picked up from the DTA traces, with the number of phases identified from XRPD analysis. The main phases identified from XRPD were NaCa(PO3)3, CaP2O6 and NaFeP2O7. Using network connectivity (NC), predictions on Qn species present within the compositions investigated were made. The predicted species (metaphosphates) matched with phases identified from XRPD analysis. A decrease in dissolution rates for the bulk glass and glass fibres was seen with an increase in CaO mol%, along with an increase in Fe2O3 mol%. An increase in fibre dissolution rates was seen with a decrease in diameter size. The biocompatibility studies were conducted using a conditionally immortal muscle precursor cell line derived from the H-2Kb-tsA58 immortomouse. It was found that iron-phosphate glass fibres containing 4-5mol% Fe2O3 was sufficient for cell attachment and differentiation. It was seen that myotubes formed along the axis of the fibres (which was indicative of differentiation). The biocompatibility of these compositions was attributed to the enhanced chemical durability of the glass fibres.

Damage to peripheral nerves induces ectopic firing in sensory neurons, which can contribute to neuropathic pain. As most of the information on this topic is on dorsal root ganglia we decided to examine the influence of infra-orbital nerve section on cells of murine trigeminal ganglia. We characterized the electrophysiological properties of neurons with intracellular electrodes. Changes in the coupling of satellite glial cells (SGCs) were monitored by intracelluar injection of the fluorescent dye Lucifer yellow. Electrophysiology of SGCs was studied with the patch-clamp technique. Six to eight days after axotomy, the percentage of neurons that fire spontaneously increased from 1.6 to 12.8%, the membrane depolarized from -51.1 to -45.5 mV, the percentage of cells with spontaneous potential oscillations increased from 19 to 37%, the membrane input resistance decreased from 44.4 to 39.5 MOmega, and the threshold for firing an action potential decreased from 0.61 to 0.42 nA. These changes are consistent with increased neuronal excitability. SGCs were mutually coupled around a given neuron in 21% of the cases, and to SGCs around neighboring neurons in only 4.8% of the cases. After axotomy these values increased to 37.1 and 25.8%, respectively. After axotomy the membrane resistance of SGCs decreased from 101 MOmega in controls to 40 MOmega, possibly due to increased coupling among these cells. We conclude that axotomy affects both neurons and SGCs in the trigeminal ganglion. The increased neuronal excitability and ectopic firing may play a major role in neuropathic pain.

The insulin-like growth factor I/insulin receptor substrate 1 axis controls, in a nonredundant way, approximately 50% of cell and body size in animals from Drosophila to mice and in cells in culture. Although other factors may also intervene, cell size is strongly dependent on ribosome biogenesis, which is under the control of RNA polymerase I activity. We have previously shown that insulin receptor substrate 1 (IRS-1) translocates to the nuclei and nucleoli, where it binds to the upstream binding factor (UBF) 1, a regulator of RNA polymerase I activity. Activation of UBF1 requires its phosphorylation. However, IRS-1 is not a kinase, and we searched for an intermediate kinase that can phosphorylate UBF1. We demonstrate here that IRS-1 binds also to the phosphatidylinositol 3-kinase (PI3-K) subunits in nuclear extracts, and that the p110 subunit of PI3-K directly phosphorylates and activates UBF1, an exclusively nucleolar protein. The interaction of IRS-1, PI3-K, and UBF1 in the nucleoli provides one of the mechanisms for the effects of IRS-1 on cell and body size.

The amino acid analogue 3-nitrotyrosine (3-NT) is formed in neural cells as a result of the intense stimulation of NMDA glutamate receptors. 3-NT is involved in the pathology of diverse neurodegenerative disorders. The aim of our work is to investigate the sensitivity of cultured neural and glial cells to 3-NT. We report the morphological changes detected on mouse neuroblastoma (C1300) and rat glioma (C6) cell lines cultured in a medium supplemented with different 3-NT concentrations. Western blot displayed a selective incorporation of 3-NT into a single protein that co-migrated with tubulin. Both cell lines showed morphological changes, nuclear suffering, decreased viability and growth inhibition (starting from 90 and 360 microM for C1300 and C6, respectively). Such effects were dose-dependent, though glioma cells showed severe alterations at higher 3-NT concentrations. Our results point out a higher 3-NT sensitivity in the neural cells studied in comparison with those of glial origin. The dramatic toxicity of 3-NT in neural cells suggests further investigations focused on the biochemical mechanisms at the roots of neurodegenerative diseases.

The distribution and geometry of the dendritic trees of spinal motoneurons obey several well-established rules. Some of these rules are based on systematic relationships between quantitative geometrical features (e.g., total dendritic length) and the three-dimensional trajectory followed by dendrites from their origin to their termination. Because dendritic geometry partially determines the transmission of current and voltage signals generated by synapses on the dendritic tree, our goal was to compare the efficacy of signal transmission by dendritic trajectories that followed different directions. To achieve this goal, we constructed detailed compartmental models of the dendritic trees of three intracellularly stained biventer cervicis/complexus (BCCM) motoneurons and calculated the electronic properties of 361 dendritic paths. Each trajectory was classified according to its orientation, e.g., rostral, rostral-dorsal-lateral. The attenuation of current and voltage signals en route to the soma was strongly related to trajectory orientation. Trajectories with similar attenuation factors formed functional subunits that were arranged in distinct domains within the ventral horn. Changes in R(m) or R(i) had little effect on which trajectories belonged to each functional subunit. However, differences in the efficacy of signal transmission between subunits increased during high network activity (mimicked by decreases in R(m)). The most efficient subunit delivered two times more current and four times more voltage to the soma than the least efficient subunit. These results indicate that the input-output properties of motoneurons depend on the direction of the path taken by dendrites from their origin at the cell body to their terminals., (Copyright 2004 Wiley-Liss, Inc.)