Temporal Association Between Ischemic Muscle Perfusion Recovery and the Restoration of Muscle Contractile Function After Hindlimb Ischemia.

During incomplete skeletal muscle recovery from ischemia, such as that occurs with critical limb ischemia, the temporal relationship between recovery of muscle capillary perfusion and contractile function is poorly defined. We examined this relationship in BALB/cJ mice (N = 24) following unilateral hindlimb ischemia (HLI), which pre-clinically mimics the myopathy observed in critical limb ischemia patients. Specifically, we examined this relationship in two phenotypically distinct muscles (i.e., "oxidative" soleus - Sol and "glycolytic" extensor digitorum longus - EDL) 14- or 56-days after HLI. Although overall limb blood flow (LDPI) reached its' recovery peak (48% of control) by HLI d14, the capillary networks in both the Sol and EDL (whole mount confocal imaging) were disrupted and competent muscle capillary perfusion (perfused lectin+µm2/muscle µm2) remained reduced. Interestingly, both Sol and EDL muscles recovered their distinct capillary structures and perfusion (Con Sol; 0.056 ± 0.02 lectin+µm2/muscle µm2, and Con EDL; 0.039 ± 0.005 lectin+µm2/muscle µm2) by HLI d56 (Sol; 0.062 ± 0.011 lectin+µm2/muscle µm2 and EDL; 0.0035 ± 0.005 lectin+µm2/muscle µm2), despite no further improvement in limb blood flow (LDPI). Both muscles suffered severe myopathy, indicated by loss of dystrophin positive immunostaining and the absence of stimulation induced isometric force production at HLI d14. Dystrophin immunofluorescence returned at HLI d56, although neither myofiber CSA (µm2) nor isometric force production (58 and 28% sustained deficits, Sol and EDL, respectively) recovered completely in either muscle. In summary, we reveal that the temporal relationship between the restoration of muscle capillary perfusion and functional ischemic skeletal muscle regeneration favors competent muscle capillary perfusion recovery in BALB/c mice in a phenotypically non-distinct manner.

Measurement system for adhesion force on single particles with microelectromechanical-based actuated tweezers.

A system for measuring the adhesion force of a single particle using microelectromechanical-system-based actuated tweezers (nanotweezers) and an atomic force microscope (AFM) cantilever was developed. In the proposed technique, a particle picked up with nanotweezers is brought into contact with and separated from the cantilever. The adhesion force is determined by measuring the deflection of the cantilever at the instant of separation from the particle. The throughput of measurement is much improved compared with that of a colloid probe AFM because the particle is picked up and held only by gripping with the nanotweezers, rather than sample preparation by manual cantilever mounting. A measurement apparatus was designed to realize the proposed system, and a force-displacement curve was successfully obtained. In addition, decreases in the adhesion force due to external coating added to particles were measured using the prototype apparatus.

The convergence of regenerative medicine and rehabilitation: federal perspectives.

Regenerative rehabilitation is the synergistic integration of principles and approaches from the regenerative medicine and rehabilitation fields, with the goal of optimizing form and function as well as patient independence. Regenerative medicine approaches for repairing or replacing damaged tissue or whole organs vary from utilizing cells (e.g., stem cells), to biologics (e.g., growth factors), to approaches using biomaterials and scaffolds, to any combination of these. While regenerative medicine offers tremendous clinical promise, regenerative rehabilitation offers the opportunity to positively influence regenerative medicine by inclusion of principles from rehabilitation sciences. Regenerative medicine by itself may not be sufficient to ensure successful translation into improving the function of those in the most need. Conversely, with a better understanding of regenerative medicine principals, rehabilitation researchers can better tailor rehabilitation efforts to accommodate and maximize the potential of regenerative approaches. Regenerative rehabilitative strategies can include activity-mediated plasticity, exercise dosing, electrical stimulation, and nutritional enhancers. Critical barriers in translating regenerative medicine techniques into humans may be difficult to overcome if preclinical studies do not consider outcomes that typically fall in the rehabilitation research domain, such as function, range of motion, sensation, and pain. The authors believe that encouraging clinicians and researchers from multiple disciplines to work collaboratively and synergistically will maximize restoration of function and quality of life for disabled and/or injured patients, including U.S. Veterans and Military Service Members (MSMs). Federal Government agencies have been investing in research and clinical care efforts focused on regenerative medicine (NIH, NSF, VA, and DoD), rehabilitation sciences (VA, NIH, NSF, DoD) and, more recently, regenerative rehabilitation (NIH and VA). As science advances and technology matures, researchers need to consider the integrative approach of regenerative rehabilitation to maximize the outcome to fully restore the function of patients.

Body weight reduction in rats by oral treatment with zinc plus cyclo-(His-Pro).

BACKGROUND AND PURPOSE: We have previously shown that treatment with zinc plus cyclo-(His-Pro) (CHP) significantly stimulated synthesis of the insulin degrading enzyme and lowered plasma insulin and blood glucose levels, alongside improving oral glucose tolerance in genetically type 2 diabetic Goto-Kakizaki (G-K) rats and in aged obese Sprague-Dawley (S-D) rats. Thus, we postulated that zinc plus CHP (ZC) treatment might also improve body weight control in these rats. We therefore determined the effects of ZC treatment on body weights in both genetically diabetic, mature G-K rats and non-diabetic, obese S-D rats. EXPERIMENTAL APPROACH: G-K rats aged 1.5-10 months and non-diabetic overweight or obese S-D rats aged 6-18 months were treated with 0-6 mg CHP plus 0-10 mg zinc L(-1) drinking water for 2-4 weeks, and changes in weight, serum leptin and adiponectin levels, food and water intakes were measured. KEY RESULTS: The optimal dose of CHP (in combination with zinc) to reduce weight and plasma leptin levels and to increase plasma adiponectin levels was close to 0.1 mg kg(-1) day(-1), in either mature G-K rats and aged overweight or obese S-D rats. Food and water intake significantly decreased in ZC treated rats in both aged S-D rats and mature G-K rats, but not in young S-D and G-K rats. CONCLUSIONS AND IMPLICATIONS: ZC treatment improved weight control and may be a possible treatment for overweight and obesity.

Bacterium organizes hierarchical amorphous structure in microbial cellulose.

A pellicle, a gel film of microbial cellulose, is a supermolecular system containing 99% of water by weight, which is closely related to an amorphous structure in it. Using ultra-small-angle neutron scattering, in order to cover over a wide range of length scales from nm to 10 microm, we examined the hierarchical amorphous structure in the microbial cellulose, which is synthesized by a bacterium (Acetobacter xylinum). The microbial cellulose swollen by water shows small-angle scattering that obeys a power law q -behavior according to q -alpha as a function of the magnitude of the scattering vector q . The power law, determined by scattering, is attributed to a mass fractal due to the distribution of the center of mass for the crystallite (microfibril) in amorphous cellulose swollen by water. As q increases, alpha takes the values of 2.5, 1, and 2.35, corresponding, respectively, to a gel network composed of bundles, a bundle composed of cellulose ribbons, and concentration fluctuations in a bundle. From the mass fractal q -behavior and its length scale limits, we evaluated a volume fraction of crystallite in microbial cellulose. It was found that 90% of the cellulose bundle is occupied by amorphous cellulose containing water.

Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage.

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.

Dynamic analysis of coagulation of low turbidity water sources using Al- and Fe-based coagulants.

The direct filtration system is widely used in the treatment of source waters with low and stable turbidity. We have previously indicated the importance of optimizing agitation strength GR and time TR in rapid mixing tanks in order to decrease filter head loss and treated water turbidity in direct filtration. In the present study, we employ a batch-type coagulation experimental apparatus that incorporates a high-sensitivity particle counter, where the particulate concentrations are measured continuously after injection of coagulant, in order to clarify the fundamental coagulation and microfloc formation dynamics. Specifically, it is shown that, after injection of the coagulant, coagulation and microfloc formation occur through distinct periods: an agglomeration preparation period, followed by an agglomeration progression period, and then finally an agglomeration stabilization period, and that optimization of the GR value is the most important consideration, although both the coagulant concentration and GR influence the time at which agglomeration begins in the preparatory period, the time at which agglomeration stabilizes after the progression period, and the concentration of initial particles with diameters of 1-3 microm at completion of agglomeration.

Mutans streptococci dose response to xylitol chewing gum.

Xylitol is promoted in caries-preventive strategies, yet its effective dose range is unclear. This study determined the dose-response of mutans streptococci in plaque and unstimulated saliva to xylitol gum. Participants (n = 132) were randomized: controls (G1) (sorbitol/maltitol), or combinations giving xylitol 3.44 g/day (G2), 6.88 g/day (G3), or 10.32 g/day (G4). Groups chewed 3 pellets/4 times/d. Samples were taken at baseline, 5 wks, and 6 mos, and were cultured on modified Mitis Salivarius agar for mutans streptococci and on blood agar for total culturable flora. At 5 wks, mutans streptococci levels in plaque were 10x lower than baseline in G3 and G4 (P = 0.007/0.003). There were no differences in saliva. At 6 mos, mutans streptococci in plaque for G3 and G4 remained 10x lower than baseline (P = 0.007/0.04). Saliva for G3 and G4 was lower than baseline by 8 to 9x (P = 0.011/0.038). Xylitol at 6.44 g/day and 10.32 g/day reduces mutans streptococci in plaque at 5 wks, and in plaque and unstimulated saliva at 6 mos. A plateau effect is suggested between 6.44 g and 10.32 g xylitol/day.

Bone morphogenetic protein 4 promotes pulmonary vascular remodeling in hypoxic pulmonary hypertension.

We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4(lacZ/+) mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.

Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, in periodontal diseased tissue.

The regulatory role of chemokines and chemokine receptors on specific leucocyte recruitment into periodontal diseased tissue is poorly characterized. We observed that leucocytes infiltrating inflamed gingival tissue expressed marked levels of CX3CR1. In periodontal diseased tissue, the expression of fractalkine and CX3CR1 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and further, fractalkine was distributed mainly on endothelial cells, as shown by immunohistochemistry. Moreover, we can detect CX3CR1-expressing cells infiltrated in periodontal diseased tissue by immunohistochemical staining. Furthermore, fractalkine production by human umbilical vein endothelial cells (HUVEC) was up-regulated by pathogen-associated molecular patterns (PAMPs), including Porphyromonas gingivalis lipopolysaccharide (LPS). Thus, these findings suggested that CX3CR1 and the corresponding chemokine, fractalkine may have an important regulatory role on specific leucocyte migration into inflamed periodontal tissue.

KC chemokine expression by TGF-beta in C3H10T1/2 cells induced towards osteoblasts.

The effects of TGF-beta on expression of the platelet-derived growth factor-induced KC protein were explored in mouse mesenchymal C3H10T1/2 and pre-osteoblastic MC3T3-E1 cells to identify a potential role for TGF-beta in expression of angiogenic cytokines during osteogenic differentiation. KC is a member of the CXC chemokine family with homology to human IL-8, a potent neutrophilic chemotactic cytokine. TGF-beta treatment results in increased KC mRNA and protein secretion in C3H10T1/2 induced towards the osteoblastic lineage with all-trans-retinoic acid. This is due to up-regulated transcription rather than enhanced mRNA stability. No induction of KC expression was seen in untreated C3H10T1/2 or MC3T3-E1 upon TGF-beta stimulation. Use of the translational inhibitor cycloheximide results in mRNA "superinduction" suggesting other factors are involved that normally function to down-regulate KC expression. TGF-beta-stimulated conditioned media were a potent chemostimulant for human microvascular endothelial cells (HMEC-1). This activity could be inhibited by pre-incubation with anti-KC neutralizing antibodies.

Serial passage of MC3T3-E1 cells down-regulates proliferation during osteogenesis in vitro.

Generally, fibroblast-like cells and other types of human cells have been used to demonstrate the principles of replicative senescence in vitro and in vivo. These cells go through three stages of proliferation, including vigorous proliferation, declining proliferation and quiescence or no proliferation. Any variation of this process occurring in osteoprogenitor cells may offer insight into the mechanism of age-related osteopaenia that predisposes individuals to osteoporosis and bone fractures. We selected MC3T3-E1 cells derived from mouse calvaria to study the mechanism of replicative senescence of pre-osteogenic cells because: (i) these cells constitute a well-known model for studying osteogenesis in vitro; (ii) they undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture; and (iii) they show signs of replicative senescence. These cells were aged by multiple passaging before their use for studying growth kinetics and the effects of population density, effect of extracellular matrix (ECM), size and phases of the cell cycle. Our results show that (i) MC3T3-E1 cells go through the first two stages of proliferation in a manner similar to human cells, but escape the quiescent phase; (ii) the rate of proliferation is similar for low passage (LP) and high passage (HP) cells, but is decreased in very high passage cells (VHP); (iii) growth inhibition is observed using HP cells seeded at high density; (iv) HP ECM stimulates proliferation of both LP and HP cells; (v) a small increase in cell size is observed in HP cells, but no change is seen in the distribution analysis of their cell cycle; (vi) distribution analysis of the cell cycle of VHP cells reveals a decreased and an increased frequency of cells in S and G2 + M phases of their cell cycle, respectively. These results suggest that the mouse MC3T3-E1 cell line exhibits many of the cellular and molecular markers associated with replicative senescence in culture as defined by human cells, such as fibroblast-like cells. Alteration in the sensitivity of MC3T3-E1 cells to intercellular contact and increase in cell size are the primary factors contributing to decreased proliferation of HP cells.

Macrophage inflammatory protein 3alpha-CC chemokine receptor 6 interactions play an important role in CD4+ T-cell accumulation in periodontal diseased tissue.

The regulatory role of chemokines and chemokine receptors on specific lymphocyte recruitment into periodontal diseased tissue is poorly characterized. We observed that lymphocytes infiltrating inflamed gingival tissue expressed marked levels of CCR6. In periodontal diseased tissue, the expression of MIP-3alpha mRNA was detected by RT-PCR and further, MIP-3alpha was distributed in the basal layer of gingival epithelial cells, microvascular endothelial cells and the areas of inflammatory cells as shown by immunohistochemistry. Moreover, CCR6-expressing cells infiltrated into periodontal diseased tissue, and the proportion of CCR6-positive CD4+ T cells was significantly elevated in periodontal diseased tissue compared with peripheral blood in the same patients. Furthermore, gingival lymphocytes isolated from patients showed migration toward MIP-3alpha in an in vitro chemotaxis assay in which migration was abrogated by specific antibody to CCR6. Thus, these findings suggested that CCR6 and the corresponding chemokine, MIP-3alpha may have an important regulatory role in specific lymphocyte migration into inflamed periodontal tissue.

Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro.

Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l-lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l-lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation.

Morphologic and molecular evidence for gap junctions and connexin 43 and 45 expression in annulus fibrosus cells from the human intervertebral disc.

Data are presented which provide evidence for gap junction formation and connexin (Cx) 43 and 45 gene expression in human intervertebral disc cells in vivo and in vitro. These findings in cells from the annulus are important in conjunction with the well-recognized loss of disc cells during aging and disc degeneration. As a result of this loss of cells, cell-cell communication, which we propose is an important, but as yet poorly understood, mechanism which links and coordinates cellular function throughout the entire population of disc cells, is also disrupted. These studies provide additional information on the fundamental cell biology of the disc cell and provide an additional framework for understanding aging, degeneration and potential repair of the human disc.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGF-beta1) alter connexin 43 phosphorylation in MC3T3-E1 Cells.

BACKGROUND: Bone morphogenetic proteins (BMPs) and transforming growth factor-betas (TGF-betas) are important regulators of bone repair and regeneration. BMP-2 and TGF-beta1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-beta1 to investigate a possible mechanism for the inhibition of GJIC. RESULTS: Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-beta1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. CONCLUSIONS: BMP-2 and TGF-beta1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-beta1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.

Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.

Effect of serial passage on gene expression in MC3T3-E1 preosteoblastic cells: a microarray study.

The osteoblastic function of mouse preosteoblastic MC3T3-E1 cells, as measured by alkaline phosphatase activity and osteocalcin secretion, decreases after serial passage. To uncover genes responsible for decreased osteoblastic function in high-passage cells, we have studied passage-dependent change of gene expression in MC3T3-E1 cells. Changes in the expression pattern of 2000 selected genes were examined simultaneously by comparing mRNA levels between MC3T3-E1 cells at passage 20 and passage 60 using the cDNA microarray analysis. Significant changes in the steady-state abundance of 27 mRNAs were observed in response to different passage numbers, including 17 known genes, 4 ESTs with homology to known genes, and 6 genes with no previously described function or homology. Northern blot analysis was used to verify and quantify the expression of selected genes, and revealed a significant higher level of up- and down-regulation compared to microarray data. These results indicate the existence of a significant change in gene expression in osteoblastic cells undergoing serial passages. Such changes might be responsible for a reduction in bone regeneration in older osteoblasts. Potential roles of selected genes in bone aging are discussed.

HUNT: launch of a full-length cDNA database from the Helix Research Institute.

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.