Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

Identification of residues and domains of Raf important for function in vivo and in vitro

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.

Unexpected clobetasol propionate profile in human stratum corneum after topical application in vitro

Purpose. The validity of using drug amount-depth profiles in stratum corneum to predict uptake of clobetasol propionate into stratum corneum and its transport into deeper skin layers was investigated. Methods. In vitro diffusion experiments through human epidermis were carried out using Franz-type glass diffusion cells. A saturated solution of clobetasol propionate in 20% (V/V) aqueous propylene glycol was topically applied for 48 h. Steady state flux was calculated from the cumulative amount of drug permeated vs. time profile. Epidermal partitioning was conducted by applying a saturated drug solution to both sides of the epidermis and allowing time to equilibrate. The tape stripping technique was used to define drug concentration-depth profiles in stratum corneum for both the diffusion and equilibrium experiments. Results. The concentration-depth profile of clobetasol propionate in stratum corneum for the diffusion experiment is biphasic. A logarithmic decline of the drug concentration over the first four to five tape strips flattens to a relatively constant low concentration level in deeper layers. The drug concentration-depth profile for the equilibrium studies displays a similar shape. Conclusions. The shape of the concentration-depth profile of clobetasol propionate is mainly because of the variable partitioning coefficient in different stratum corneum layers.

The synthesis and X-ray crystal structure of 9-carboxyhexahydro-7-methoxy-4a,7-ethano-benzopyran-5-en-1-one

9-Carboxyhexahydro-7-methoxy-4a,7-ethano-benzopyran-5-en-1-one (1) was prepared and examined by X-ray crystallography to probe its potential as a new peptide scaffold/template. The crystal structure of the anhydride precursor 7-(2-acetoxyethyl)-4-methoxy-3a,4,7,7a-tetrahydro-4,7-ethanoisobenzofuran-1,3-dione (6) is also reported.

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast-mineralized matrix complex. Histologically, at this time point a tissue structure with a connective tissue-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of inummodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Antiproliferative and phenotype-transforming antitumor agents derived from cysteine

Selective destruction of malignant tumor cells without damaging normal cells is an important goal for cancer chemotherapy in the 21st century. Differentiating agents that transform cancer cells to either a nonproliferating or normal phenotype could potentially be tissue-specific and avoid side effects of current drugs. However, most compounds that are presently known to differentiate cancer cells are histone deacetylase inhibitors that are of low potency or suffer from low bioavailability, rapid metabolism, reversible differentiation, and nonselectivity for cancer cells over normal cells. Here we describe 36 nonpeptidic compounds derived from a simple cysteine scaffold, fused at the C-terminus to benzylamine, at the N-terminus to a small library of carboxylic acids, and at the S-terminus to 4-butanoyl hydroxamate. Six compounds were cytotoxic at nanomolar concentrations against a particularly aggressive human melanoma cell line (MM96L), four compounds showed selectivities of greater than or equal to5:1 for human melanoma over normal human cells (NFF), and four of the most potent compounds were further tested and found to be cytotoxic for six other human cancer cell lines (melanomas SK-MEL-28, DO4; prostate DU145; breast MCF-7; ovarian JAM, CI80-13S). The most active compounds typically caused hyperacetylation of histones, induced p21 expression, and reverted phenotype of surviving tumor cells to a normal morphology. Only one compound was given orally at 5 mg/kg to healthy rats to look for bioavailaiblity, and it showed reasonably high levels in plasma (C-max 6 mug/mL, T-max 15 min) for at least 4 h. Results are sufficiently promising to support further work on refining this and related classes of compounds to an orally active, more tumor-selective, antitumor drug.

Potent cyclic antagonists of the complement C5a receptor on human polymorphonulcear leukocytes. Relationships between structures and activity

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC50, 20 nM(-1) muM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Gelatinisation of starch in mixtures of sugars. II. Application of differential scanning calorimetry

Differential scanning calorimetry was used to investigate the effect of mixtures of glucose and fructose, and five types of honeys on starch gelatinisation. At a 1:1 starch:water ratio, glucose generally increased the enthalpy (DeltaH(gel)) and temperatures (T-onset, T-peak and T-end) of gelatinisation more than fructose. Upon mixing, DeltaH(gel) of the low-temperature endotherm decreased in comparison to the sole sugars, but was fairly constant (7.7 +/- 0.33 J/g dry starch). DeltaH(gel) of the high-temperature endotherm increased with the fructose content. For both endotherms, the gelatinisation temperatures were unchanged (CV less than or equal to 3%) for the mixtures. With the honeys (moisture, 14.9-18.0%; fructose, 37.2-44.0%; glucose, 28.3-31.9%) added at 1.1-4.4 g per g dry starch, the enthalpy and temperatures of gelatinisation did not vary significantly (CV less than or equal to 6%). Typical thermograms are presented, and the results are interpreted in the light of the various proposed mechanisms for starch gelatinisation in sugar-water systems, total sugar content and possible sugar-sugar interactions. The thermograms were broader in the presence of the sugars and honeys, and a biphasic character was consistently exhibited. The application of an exponential equation to the gelatinisation temperatures of the starch-honey mixtures revealed an opposing influence of fructose and glucose during gelatinisation. The mechanism of starch gelatinisation may be better understood if techniques could be perfected to quantify breakage and formation of hydrogen bonds in the starch granules, and suggested techniques are discussed. (C) 2004 Elsevier Ltd. All rights reserved.

The effect of timing and severity of water deficit on growth, development, yield accumulation and nitrogen fixation of mungbean

Mungbean (Vigna radiata L.), as a dryland grain legume, is exposed to varying timing and severity of water deficit, which results in variability in grain yield, nitrogen accumulation and grain quality. In this field study, mungbean crops were exposed to varying timing and severity of water deficit in order to examine: (1) contribution of the second flush of pods to final grain yield with variable timing of relief from water deficit, (2) the sensitivity to water deficit of the accumulation of biomass and nitrogen (N) and its partitioning to grain, and (3) how the timing of water deficit affects the pattern of harvest index (HI) increase through pod filling. The results showed that the contribution of the second flush to final yield is highly variable (1-56%) and can be considerable, especially where mid-season stress is relieved at early pod filling. The capacity to produce a second flush of pods did not compensate fully for yield reduction due to water stress. Relief from mid-season stress also resulted in continued leaf production, N-2 fixation and vegetative biomass accumulation during pod filling. Despite the wide variation in the degree of change in vegetative biomass and N during pod filling, there were strong relationships between grain yield and net-above-ground biomass at maturity, and grain N and above-ground N at maturity. Only in the extreme situations were HI and nitrogen HI affected noticeably. In those treatments where there was a large second flush of pods, there was a pronounced biphasic pattern to pod number production, with HI also progressing through two distinct phases of increase separated by a plateau. The proportion of grain yield contributed to by biomass produced before pod filling varied from 0 to 61% with the contribution greatest under terminal water deficit. There was a larger effect of water deficit on N accumulation, and hence N-2 fixation, than on biomass accumulation. The study confirmed the applicability of a number of long-standing physiological concepts to the analysis of the effect of water deficit on mungbean, but also highlighted the difficulty of accounting for timing effects of water deficit where second flushes of pods alter canopy development, biomass and yield accumulation, and N dynamics. Crown Copyright (C) 2003 Published by Elsevier B.V. All rights reserved.

Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons

1 The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2 Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. 3 Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. 4 Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced < 50% by CTX GVIA 10-100 nM. 5 Capsaicin (3 muM) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6 The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 muM) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 muM) reduced but did not abolish the remaining neurogenic dilations. 7 Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

Scaffolds, stem cells, and tissue engineering: A potent combination!

Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.

Changes in three dimensional lumbo-pelvic kinematics and trunk muscle activity with speed and mode of locomotion

Background Control of the trunk is critical for locomotor efficiency. However, investigations of trunk muscle activity and three-dimensional lumbo-pelvic kinematics during walking and running remain scarce. Methods. Gait parameters and three-dimensional lumbo-pelvic kinematics were recorded in seven subjects. Electromyography recordings of abdominal and paraspinal muscles were made using fine-wire and surface electrodes as subjects walked on a treadmill at 1 and 2 ms(-1) and ran at 2, 3, 4 and 5 ms(-1). Findings. Kinematic data indicate that the amplitude but not timing of lumbo-pelvic motion changes with locomotor speed. Conversely, a change in locomotor mode is associated with temporal but not spatial adaptation in neuromotor strategy. That is, peak transverse plane lumbo-pelvic rotation occurs at foot strike during walking but prior to foot strike during running. Despite this temporal change, there is a strong correlation between the amplitude of transverse plane lumbo-pelvic rotation and stride length during walking and running. In addition, Jumbo-pelvic motion was asymmetrical during all locomotor tasks. Trunk muscle electromyography occurred biphasically in association with foot strike. Transversus abdominis was tonically active with biphasic modulation. Consistent with the kinematic data, electromyography activity of the abdominal muscles and the superficial fibres of multifidus increased with locomotor speed, and timing of peak activity of superficial multifidus and obliquus externus abdominis was modified in association with the temporal adaptation in lumbo-pelvic motion with changes in locomotor mode. Interpretation. These data provide evidence of the association between lumbo-pelvic motion and trunk muscle activity during locomotion at different speeds and modes. (c) 2005 Elsevier Ltd. All rights reserved.

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production

Introduction: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. Materials and Methods: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappa B and activator protein-1 (AP-1) activity, Western blotting for phosphoextracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. Results: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappa B activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. Conclusion: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Subcellular localization determines MAP kinase signal output

The Raf-MEK-ERK MAP kinase cascade transmits signals from activated receptors into the cell to regulate proliferation and differentiation. The cascade is controlled by the Ras GTPase, which recruits Raf from the cytosol to the plasma membrane for activation. In turn, MEK, ERK, and scaffold proteins translocate to the plasma membrane for activation. Here, we examine the input-output properties of the Raf-MEK-ERK MAP kinase module in mammalian cells activated in different cellular contexts. We show that the MAP kinase module operates as a molecular switch in vivo but that the input sensitivity of the module is determined by subcellular location. Signal output from the module is sensitive to low-level input only when it is activated at the plasma membrane. This is because the threshold for activation is low at the plasma membrane, whereas the threshold for activation is high in the cytosol. Thus, the circuit configuration of the module at the plasma membrane generates maximal outputs from low-level analog inputs, allowing cells to process and respond appropriately to physiological stimuli. These results reveal the engineering logic behind the recruitment of elements of the module from the cytosol to the membrane for activation.