The Cytoskeleton Regulates Cell Attachment Strength

Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature—only during shear application—also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.     

Specific energy requirement for compacting corn stover

Corn stover is a major crop residue for biomass conversion to produce chemicals and fuels. One of the problems associated with the supply of corn stover to conversion plants is the delivery of feedstock at a low cost. Corn stover has low bulk density and it is difficult to handle. In this study, chopped corn stover samples were compacted in a piston cylinder under three pressure levels (5, 10, 15 MPa) and at three moisture content levels (5%, 10%, 15% (wb)) to produce briquettes. The total energy requirement to compress and extrude briquette ranged from 12 to 30 MJ/t. The briquette density ranged from 650 to 950 kg/m3 increasing with pressure. Moisture content had also a significant effect on briquette density, durability and stability. Low moisture stover (5-10%) resulted in denser, more stable and more durable briquettes than high moisture stover (15%).     

Attachment of Entamoeba histolytica to glass in a defined maintenance medium: specific requirement for cysteine and ascorbic acid

Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12-24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N2 atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.     

Continual Cell Deformation Induced via Attachment to Oriented Fibers Enhances Fibroblast Cell Migration

Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm for maximal contact with the fiber edge. In the case of en-mass cell migration out of an agarose droplet, fibroblasts on flat surfaces emerged with an enhanced velocity, v = 52μm/h, that decreases to the single cell value, v = 28μm/h within 24 hours and remained constant for at least four days. Fibroblasts emerging on fibrillar surfaces emerged with the single cell velocity, which remained constant for the first 24 hours and then increased reaching a plateau with more than twice the initial velocity within the next three days. The focal adhesions were distributed uniformly in cells on flat surfaces, while on the fibrillar surface they were clustered along the cell periphery. Furthermore, the number of focal adhesions for the cells on the flat surfaces remained constant, while it decreased on the fibrillar surface during the next three days. The deformation of the cell nuclei was found to be 50% larger on the fiber surfaces for the first 24 hours. While the mean deformation remained constant on the flat surface, it increased for the next three days by 24% in cells on fibers. On the fourth day, large actin/myosin fibers formed in cells on fibrillar surfaces only and coincided with a change from the standard migration mechanism involving extension of lamellipodia, and retraction of the rear, to one involving strong contractions oriented along the fibers and centered about the nucleus.     

Attachment of Flagellar Basal Bodies to the Cell Envelope: Specific Attachment to the Outer, Lipopolysaccharide Membrane and the Cytoplasmic Membrane

A procedure is described for the purification of the Escherichia coli outer membrane (lipopolysaccharide or L membrane) with flagella still attached. The resulting lipopolysaccharide membrane was in the form of vesicles that had a trilaminar structure in thin section and contained about 55% lipopolysaccharide and 45% protein. T2 or T4 phage preadsorbed to E. coli were found attached to the purified lipopolysaccharide membrane. Flagella were bound to the purified lipopolysaccharide membrane specifically at the basal body ring closest to the hook (the L ring). The cytoplasmic membrane in preparations from osmotically lysed E. coli spheroplasts or Bacillus subtilis protoplasts was specifically attached to flagella at the basal body ring farthest from the hook (the M ring). In the E. coli preparation, lipopolysaccharide membrane was also present and was attached to the L ring. From these data and a knowledge of the structure and dimensions of the E. coli flagellar basal body and cell envelope, a model for flagellar attachment is deduced.     

促细胞黏附生长的自组装肽水凝胶

随着细胞与组织工程的迅猛发展,能够促进细胞黏附、生长和分化的生物材料基质支架的研究日益重要。具有生物相容性且含水量超过99％的自组装肽水凝胶因其很好地符合理想的生物材料基质支架标准而备受重视。这类自我互补的两亲寡肽含50％的带电残基,并且以交替的离子亲水性和不带电的氨基酸残基周期性重复为特征;在其寡肽的氨基末端可用直接固相合成法修饰几个短序列生物活性模体进行功能化,用以促进不同细胞的黏附生长和靶向定位。现对自组装肽水凝胶的结构特征、自组装机制、对细胞黏附生长的影响以及未来自组装肽生物材料设计的目标进行综述．     

Tamoxifen Inhibition of Ocular Melanoma Cell Attachment to Matrix Proteins

Tamoxifen plays a major role in the management of breast cancer in women and is currently used to a lesser extent in other neoplasias. Many of the pharmacological properties of tamoxifen are consistent with anti-estrogen activity, but it also has significant, although lesser, benefit in patients whose tumours are estrogen-receptor negative. We recently reported that murine B16 melanoma cell attachment to extracellular matrix proteins can be inhibited by calmodulin antagonists. In seeking a calmodulin antagonist that could be used clinically, we investigated tamoxifen, which is known to have calmodulin antagonist activity in vitro, and confirmed that it will inhibit murine melanoma cell attachment in vitro. In the current study, we examined the effect of tamoxifen on the attachment of human ocular melanoma cell lines to a range of extracellular matrix substrates to evaluate the potential relevance of calmodulin antagonists, including tamoxifen, to reducing metastatic spread of these tumours. We report that six ocular melanoma cell lines established from choroidal melanoma tumours showed rapid attachment to a range of substrates and that this attachment can be significantly reduced by an experimental calmodulin antagonist (J8) and by tamoxifen. In summary, we conclude that the ability of calmodulin antagonists, including tamoxifen, to inhibit ocular melanoma cell attachment to matrix proteins in vitro merits further investigation as it may offer another approach to reducing metastatic spread of these tumours.     

The requirement for energy transducing ATPase for anaerobic motility in Escherichia coli

A sensitive radioactive assay for motility based on infection with ³²P-labeled, flagella-specific phage x is described. Anaerobic infection with phage x, which requires a motile host, is blocked in energy transducing ATPase (unc A) mutants. Anaerobic infection is restored by addition of NO₃⁻ which functions as a terminal electron acceptor for anaerobic respiration.     

小鼠FAAP蛋白对细胞黏附的影响

黏着斑相关蛋白(focal adhesion associated protein,FAAP)由小鼠D10Wsu52e基因编码,在进化上十分保守,但该蛋白质的生物学功能并不清楚．为此,首先通过细胞组分分离的方法研究了FAAP蛋白分布的细胞组分,结果表明FAAP主要存在于细胞质和细胞膜中．细胞蛋白表达量分析表明,细胞中FAAP与黏连蛋白受体(LR)表达量呈现正相关性．同时,细胞黏附实验表明,FAAP与LR对细胞黏附影响类似,也能够抑制细胞的黏附．这些实验结果为深入研究FAAP蛋白功能提供了依据．     

The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.     

Free energy requirement for domain movement of an enzyme

Domain movement is sometimes essential for substrate recognition by an enzyme. X-ray crystallography of aminotransferase with a series of aliphatic substrates showed that the domain movement of aspartate aminotransferase was changed dramatically from an open to a closed form by the addition of only one CH(2) to the side chain of the C4 substrate CH(3)(CH(2))C((alpha))H(NH(3)(+))COO(-). These crystallographic results and reaction kinetics (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63; Kawaguchi, S. and Kuramitsu, S. (1998) J. Biol. Chem. 273, 18353-18364) enabled us to estimate the free energy required for the domain movement.     

The energy requirement for protein synthesis in rat brain mitochondria purified by phase partition

Brain mitochondria purified by phase partition showed a higher rate of 14C-leucine incorporation into proteins with an endogenous source of ATP than with an exogenous ATP-generating system. Under the former conditions the presence of atractyloside increased the 14C-leucine incorporation into proteins. The effects of different valinomycin concentrations plus attractyloside on intramitochondrial ATP levels and 14C-leucine incorporation into proteins have been studied. The results indicate that the protein synthesis in brain mitochondria is dependent on the intramitochondrial ATP concentration.     

Women at altitude: energy requirement at 4,300 m.

To test the hypotheses that prolonged exposure to moderately high altitude increases the energy requirement of adequately fed women and that the sole cause of the increase is an elevation in basal metabolic rate (BMR), we studied 16 healthy women [21.7 +/- 0.5 (SD) yr; 167.4 +/- 1.1 cm; 62.2 +/- 1.0 kg]. Studies were conducted over 12 days at sea level (SL) and at 4,300 m [high altitude (HA)]. To test that menstrual cycle phase has an effect on energetics at HA, we monitored menstrual cycle in all women, and most women (n = 11) were studied in the same phase at SL and HA. Daily energy intake at HA was increased to respond to increases in BMR and to maintain body weight and body composition. Mean BMR for the group rose 6.9% above SL by day 3 at HA and fell to SL values by day 6. Total energy requirement remained elevated 6% at HA [ approximately 670 kJ/day (160 kcal/day) above that at SL], but the small and transient increase in BMR could not explain all of this increase, giving rise to an apparent "energy requirement excess." The transient nature of the rise in BMR may have been due to the fitness level of the subjects. The response to altitude was not affected by menstrual cycle phase. The energy requirement excess is at present unexplained.     

Cell energy metabolism: An update

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD⁺ molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect.     

Cation Type Specific Cell Remodeling Regulates Attachment Strength

Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α₅-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions.     

The Terpolymer Produced by Azotobacter Chroococcum 7B: Effect of Surface Properties on Cell Attachment

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by ¹H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.