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The distribution of essential nutrients such as potassium (K), phosphorous (P), calcium (Ca), sulfur (S) and chlorine (Cl) within root tissues is crucial aspect of plant growth but nothing is known regarding this subject in seedlings of dicotyledons such as peanut (Arachis hypogaea). We used scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) and inductively coupled plasma optical emission spectrometry (ICP-OES) to analyze element distributions in developing root tissues. Distribution patterns and concentrations of endogenous K, P, S and exogenous Ca from seeds grown in water varied along the length of peanut radicle and were tissue specific. Semi-quantitative SEM/EDS data indicated 24 mM K in the cortex and 46 mM K in the stele at a distance of 5 mm from the root tip. The P concentration in the same region was 15 mM in cortex and 22 mM in stele. The concentration of K and P decreased in the more mature parts of the root. SEM–EDS element concentrations values were lower for K and higher for P when compared with the ICP-OES data of root segments. Peanut roots grown in 10 mM Ca(NO3)2 solution for hours showed tissue-specific Ca distribution at 25 mm from the root tip with the highest levels detected in portions of the cortex. The parent root cortex centrifugal to the tip of developing lateral root primordia had lower levels of calcium than could be detected by SEM/EDS.

K, P, Cl, and Ca are distributed in tissue-specific patterns in Zea mays seedlings. These elements were mapped and analyzed using a relatively simple semi-quantitative technique, i.e., fast freezing, followed by freeze fracturing, then freeze drying, and finally scanning electron microscopy/energy-dispersive spectroscopy (SEM/EDS). In the radicle, endogenously derived (i.e., from seed) K and P transition from being homogenous in the apical meristem to tissue-specific in older regions. At 3 mm from the radicle apex, K concentration is approximately 40 mM in mid-cortex and decreases by approximately 50% at 15 mm. From 3 to 55 mm, P concentration in pericycle is approximately twice that found in adjacent regions. Ca is not detectable in younger portions of the radicle by SEM/EDS, but in older regions, it is present at 13 mM in mid-cortex. K concentration values of entire radicles analyzed with inductively coupled plasma optical emission spectrometry (ICP-OES) exceeded the SEM/EDS values. For Ca, the reverse was true. But, SEM/EDS analysis did not include several vascular tissues that contained high concentrations of K and low concentrations of Ca. The inception of lateral root primordia was accompanied by a localized decrease in Ca in cortical regions that were centrifugal to the primordium tip. A region of O-rich cells in endosperm was identified centripetal to the aleurone. These results indicate that (1) outer, mid-, and inner cortical regions, as well as the adjacent tissues, have distinct ion accumulation properties, and (2) ions are concentrated in some radicle tissues prior to development of Casparian strips.

Potassium (K), phosphorous (P), and carboxyfluorescein (CF) accumulate in functionally distinct tissues within the pine seedling root cortex. Seedlings of Pinus pinea translocate exogenous CF and endogenous K and P from the female gametophyte/cotyledons to the growing radicle. Following unloading in the root tip, these materials accumulate in characteristic spatial patterns. Transverse sections of root tips show high levels of P in a circular ring of several layers of inner cortical cells. K and CF are minimal in the high P tissue. In contrast, high levels of K and CF accumulate in outer cortical cells, and in the vascular cylinder. These patterns are a property of living tissue because they change after freeze-thaw treatment, which kills the cells and results in uniform distribution of K and P. K concentration can be reduced to undetectable levels by incubation of roots in 100 mM NaCl. Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) of root segments both reliably determine K and P concentrations.

Micro- and multiscale material properties must be considered when manufacturing miniature devices, especially when considering utilizing multiscale sheet metal hydroforming processes. One of the critical considerations during the design process is the burst pressure for the sheet metal which is a limiting factor for potential hydroforming operations. In order to simplify determining tearing or rupture conditions, it is sometimes desired to use analytical methods for estimating material properties, including burst pressures, which occur shortly after material instability. Many researchers have developed approximate methods for predicting deformation during open die hydroforming based upon analytical approaches for biaxial conditions for circular and elliptical dies. Additionally, extracting material properties of sheet metal under biaxial conditions such as bulge hydroforming more closely matches forming conditions that the sheet metal will undergo for actual parts. The objective of the current research was to evaluate the analytically developed models’ ability to predict burst conditions and compare those burst results to those obtained from finite element models and experimentation. Stainless steel (annealed 0.2-mm thick AISI 304) was hydroformed in a circular open die with diameter of 11mm. Elliptical dies were also evaluated that had minor diameters of 11mm and aspect ratios down to 0.5. It was found that using the analytical method developed specifically for circular dies was a good predictor for the burst pressure while the more general analytical method for elliptical dies did not agree with either results from finite element analysis or experimental results.

Meso- and microscale sheet metal forming represents new and attractive solutions to many manufacturing problems for product miniaturization. Larger organizations are utilizing commercially available microscale digital image correlation systems to measure the strains on these scales. The cost of these systems is preventing smaller research and development organizations from entering this challenging area or they are sacrificing the ability to determine strains and evaluate material behavior at the microscale. However, microscale strain grid measurement has the advantage over digital image correlation when the researchers wish to avoid polishing and etching the surface of the sheet metal to make the grain structure visible for digital image correlation and where tooling interferes with obtaining images of the workpiece in real time. This article evaluates the strain measurement and strains resulting from multiscale sheet metal hydroforming operations for annealed 0.2-mm-thick ASTM 304 stainless steel using a simple method for producing microscale grids that has been previously described. The gridding methodology was shown to be accurate with high repeatability. In addition, a strain grid measurement method using an optical microscope and digital camera is described and an error analysis was performed. Provided reasonable care is taken, the inherent error in undeformed parts is 0.76% of true strain for samples with 127 µm grids using the strain measurement system described. The maximum variation in the mesoscale and microscale strain measurement static bulge testing was ±2.4% and more typically ±1.3% of true strain. With care, the errors were reduced to less than 1% of strain. Microscale strains from sheet bulge hydroforming experiments for 11, 5, and 1 mm diameter dies are used to show that the strains measured are reasonable and consistent.

Graphene oxide and chitosan are promising materials for tissue regeneration. The present study explores a novel biomimetic mineralization route employing a graphene oxide (GO)-chitosan (CS) conjugate as a template material for the biomineralization of hydroxyapatite (HAP). Structural and morphological studies involving X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy indicated that extensive mineralization occurred in the CS-GO conjugate system because of strong electrostatic interactions between the functional groups (carboxyl groups of GO and amino groups of CS) and calcium ions in the simulated body fluid (SBF). The combination of chitosan-graphene oxide conjugate and biomineralization was advantageous in favorably modulating cellular activity (osteoblast functions: cell attachment, proliferation, actin, vinculin and fibronectin expression). It is concluded that biomineralized hydroxyapatite in the HAP-CS-GO system induced homogeneous spatial osteoblastic cell growth and quantitatively (e.g. area) and qualitatively (e.g. mineral-to-matrix ratio) increased mineralization in relation to the HAP-GO system. The data underscore that covalent linkage of HAP to chitosan influences osteoblastic cell differentiation, mineralization, and cell growth. The proposed system and the revelation of fundamental insights merit consideration in tissue engineering.

This paper focuses on the microstructure and fracture surface morphology of neat and carbon nanofibers (CNF) modified asphalts and hot mix asphalt (HMA) mixtures using scanning electron microscopy (SEM). Asphalt binder was modified with 1.5 % of CNF by weight of binder. The modified asphalt was used to construct HMA mixtures at various CNF dosages, mixed with aggregate, using the Superpave Gyratory compactor. Small rectangular specimens extracted from the center of large HMA samples were tested under direct tension and the fracture surface was examined under SEM. The SEM analysis developed a fundamental understanding of the role that the CNF modification plays in the performance enhancement of asphalt and HMA mixtures. It was found that CNF not only possess good adhesion characteristics but also exhibits high connectivity and were evenly distribution throughout the binder. The fracture surface morphology also revealed that CNF exhibited crack bridging at micro/nano scale which may enhance the resistance to cracking due to repeated traffic loads.

From the perspective of osseointegration, nanograined/ultrafine-grained (NG/UFG) metals provide surfaces that are different from conventional coarse-grained (CG) polycrystalline metals because of the high fraction of grain boundaries. We describe here the interplay between the cellular response and grain boundary grooving as a potential approach to enhance osteoblast functions and facilitate the biomechanical interlocking and anchorage. This is accomplished by making a relative comparison of osteoblast response of NG/UFG grains electrochemically grooved to different depths to induce different degree of nanoscale roughness with planar NG/UFG surfaces, under identical biological environment. Electrochemically grooved NG/UFG structures indicated significant attachment and proliferation, and consequently enhanced modulation of cellular response that was significantly different from planar (non-grooved) NG/UFG substrate. Consistent with cell attachment and proliferation, immunofluorescence microscopy and computational analysis indicated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on electrochemically-grooved NG/UFG substrates. These observations are indicative of accelerated response of cell–substrate interaction and activity. The behavior is attributed to average nanoscale roughness and high surface hydrophilicity of the nanoengineered surface.

Producing fuel cells bipolar plates and other devices such as microscale heat exchangers for electronics requires both macroscale and microscale forming processes. At the macroscale, typically, mechanical properties of sheet metal are determined by performing tensile tests. In addition, it has long been recognized that bi-axial tension tests, dome tests, and hydroforming or viscous bulge tests provide the basis for improved understanding of the mechanics of sheet metal forming. At the microscale strain gauges are too large for measuring strains in small regions and membrane theory is only valid at the poles of the bulge. Continuum mechanics models are useful but require tedious thickness measurements for multiple work pieces, requiring extensive sample preparation and analysis. In this paper experimental results from hydroforming tests for 0.2-mm thick annealed ASTM 304 stainless steel sheet in 11 mm, 5 mm, and 1 mm diameter open dies at various pressures were evaluated. The height of the bulge at the pole and strains based upon measurements of 127 micron strain grids were determined. These dies represent the transition from a small macroscale process to a microscale forming process. Two methods were used to estimate material properties: an analytical model and an iterative method which compared experimental strain results with the strains from a finite element model where the Holloman constitutive properties of the sheet were varied. The problems estimating material properties based upon grid strain measurement, membrane theory, and the iterative finite element approaches were investigated and the results were compared. This study indicates that membrane theory will provide adequate predictions for Holloman constructive properties provided the assumptions for membrane theory are not violated. However, using measured microscale grid deformation strains does not produce very good agreement estimates of the Holloman constitutive model when comparing experimental results with FEA strains. It is believed that while the grid strain measurement method used results in strain measurement errors of less than 1.5% of strain, this error is sufficient to result in enough uncertainty to produce results that are inconsistent with other methods.

In the context of osseointegration of metallic implants, while nanostructuring the surface favorably modulates cellular response, the disinfective attributes required during the healing process are not available. Thus, in the present study, we demonstrate that nanocrystalline titania provides cumulative benefit of enhancing osteoblasts functions to promote the efficacy of metal implants together with the disinfective attributes. To this end, the primary objective here is to examine the select functions of bone forming cells (osteoblasts) on electrocrystallized nanonodular titania-coated nanograined/ultrafine grained (NG/UFG) austenitic stainless steel. The accompanying objective is to study the disinfective/antimicrobial activity. To the best of our understanding this is the first study of nanophase titania on a non-titanium substrate. The osteoblasts functions were investigated in terms of cell attachment, proliferation, and quantitative analysis of proteins, actin and vinculin. In comparison to the bare NG/UFG substrate, the nanophase titania-coated substrate exhibited higher degree of cell attachment and proliferation which are regulated via cellular and molecular interactions with proteins, actin and vinculin. The enhanced functions of osteoblasts suggest that nanophase titania adsorbs extracellular matrix proteins, fibronectin and vitronectin from serum enhancing protein, with subsequent binding of integrins and osteoblasts precursor to titania. The antimicrobial attributes assessed in terms of degradation of methyl orange and effectiveness in killing E. coli supports the viewpoint that large surface area of titania would be instrumental in reducing the detrimental effect of biologically reactive oxygen species produced by macrophages in the vicinity of the metal bone/implant interface. In summary, the study provides some new insights concerning nanostructuring of metallic substrates with specific physical and surface properties for medical devices with significantly improved cellular response.

We describe here the structure―property―process relationship of two sets of chitosan-based scaffolds as potential materials for hard tissue bioengineering applications. The first set of scaffolds was designed to study the effect of degree of deacetylation (%DD) of chitosan at 85 and 95% DD with identical molecular weight. The second set of scaffolds were synthesized to enhance the bioactivity and mechanical properties of 95% DD chitosan scaffold by the addition of 1, 3, and 5 wt% nanohydroxyapatite (nHA). Both sets of scaffolds were processed using freezing and lyophilization and their in vitro biological response was examined using pre-osteoblast (MC 3T3-E1) cells. The pure chitosan and chitosan―nHA nanocomposite scaffolds were characterized by a three-dimensional porous structure with pore size in the range of (≈50―120 μm), irrespective of %DD and nHA content. The compression modulus of hydrated 95% DD chitosan scaffolds increased from 6.0 kPa in pure chitosan to 9.2 kPa in chitosan-5 wt% nHA. The water uptake ability of 95% DD chitosan scaffolds was lower than 85% DD chitosan scaffolds, and decreased with the addition of nHA. On the other hand the water retention ability of 95% DD chitosan scaffolds was greater than 85% DD chitosan scaffolds but did not increase much with the addition of nHA. Chitosan degrades mainly by lysozyme present in physiological fluid. After 28 days of in vitro biodegradation test with physiological fluid, the 95% DD chitosan exhibited slightly lower degradation rate as compared to 85% DD and the degradation decreased with increase in nHA content in chitosan scaffold. Pre-osteoblasts (MC3T3-E1) grown on nHA scaffolds showed improved cell attachment, more proliferation, and a more extended cellular morphology. In vitro experimental observations suggest chitosan-nHA nanocomposite scaffolds as potential biomaterials for hard tissue bioengineering (bone regeneration) or as a template for cell attachment and proliferation in the repair of osseous and chondral defects.

Given the need to develop high strength/weight ratio bioimplants with enhanced cellular response, we describe here a study focused on the processing–structure–functional property relationship in austenitic stainless steel that was processed using an ingenious phase reversion approach to obtain an nanograined/ultrafine-grained (NG/UFG) structure. The cellular activity between fibroblast and NG/UFG substrate is compared with the coarse-grained (CG) substrate. A comparative investigation of NG/UFG and CG structures illustrated that cell attachment, proliferation, viability, morphology and spread are favorably modulated and significantly different from the conventional CG structure. These observations were further confirmed by expression levels of vinculin and associated actin cytoskeleton. Immunofluorescence studies demonstrated increased vinculin concentrations associated with actin stress fibers in the outer regions of the cells and cellular extensions on NG/UFG substrate. These observations suggest enhanced cell–substrate interaction and activity. The cellular attachment response on NG/UFG substrate is attributed to grain size and hydrophilicity and is related to more open lattice in the positions of high-angle grain boundaries.

Premise of the study Primary vascular tissues of angiosperm and gymnosperm roots have significant anatomical differences. In gymnosperms, lack of protophloem sieve elements indicates a lengthy parenchymatous pathway for nutrient transport to the root apical meristem (RAM). Because F-actin is an essential component of transport in parenchyma cells, the distribution of F-actin was determined and compared among roots of several angiosperm and gymnosperm species. Methods Roots were chemically fixed and sectioned by hand to enable rapid production of many sections for labeling F-actin with phalloidin. Key results In angiosperm and gymnosperm root tips, relative intensity of F-actin labeling was highest in primary vascular tissues. Parenchyma cells in and around protophloem tended to have more F-actin while cells in cortical and protoxylem tissues tended to have less. In gymnosperms, phloem parenchyma was intensely labeled for several millimeters distal to the root apical meristem (RAM), and the F-actin is mostly composed of bundles that lie parallel to the root longitudinal axis. This orientation differed from the multidirectional arrangement of F-actin filaments in cortical cells. In angiosperms, intense F-actin labeling of pericycle and phloem parenchyma cells occurred around the first mature sieve elements. Conclusions F-actin is concentrated in the vascular cylinder, commonly in primary phloem parenchyma. In gymnosperms, the absence of sieve elements suggests that cytoplasmic streaming has a role in some aspect of phloem transport or unloading. In angiosperms, the region of intense F-actin labeling in the phloem parenchyma is limited to the extreme terminal portion of primary phloem where unloading of the earliest mature sieve elements occurs.

The objective of this study was to develop a reliable method for the measurement of pipe surface roughness. Roughness of coated pipe was measured with two linear surface profilers, a Dektak3ST® and a Hommel T 1000, and a Dimension 3000® atomic force microscope (AFM). Arithmetic roughness (Ra), root mean square roughness (Rq), and mean peak-to-valley height (RZD) were statistically analyzed. Each instrument yielded reliable results, but there were significant differences between the AFM and the other two instruments. The ability of RZD to ignore intermediate height data while focusing on extreme height data made it a useful parameter for detecting differences in height that would be the most likely to affect fluid flow in pipes.

The role and regulation of specific plant myosins in cyclosis is not well understood. In the present report, an affinity-purified antibody generated against a conserved tail region of some class XI plant myosin isoforms was used for biochemical and immunofluorescence studies of Zea mays. Myosin XI co-localized with plastids and mitochondria but not with nuclei, the Golgi apparatus, endoplasmic reticulum, or peroxisomes. This suggests that myosin XI is involved in the motility of specific organelles. Myosin XI was more than 50% co-localized with tailless complex polypeptide-1α (TCP-1α) in tissue sections of mature tissues located more than 1.0 mm from the apex, and the two proteins co-eluted from gel filtration and ion exchange columns. On Western blots, TCP-1α isoforms showed a developmental shift from the youngest 5.0 mm of the root to more mature regions that were more than 10.0 mm from the apex. This developmental shift coincided with a higher percentage of myosin XI /TCP-1α co-localization, and faster degradation of myosin XI by serine protease. Our results suggest that class XI plant myosin requires TCP-1α for regulating folding or providing protection against denaturation. Cell Motil. Cytoskeleton 57:218–232, 2004. © 2004 Wiley-Liss, Inc.

The objectives of this study were to determine: the type of coating that minimized pipe surface roughness and how the choice of metrological instrument could influence pipe surface roughness data. The internal surface of pipe was coated with either phenolic, modified novalac, epoxy, or nylon material. Roughness of coated pipe was assessed with two linear surface profilers, a Dektak3ST® and a Hommel T1000, and a Dimension 3000® atomic force microscope (AFM). Arithmetic roughness (Ra), root mean square roughness (Rq), and mean peak‐to‐valley height (RZD), were statistically analyzed. The ability of RZD to focus on the extremes of height and depth on the surface made it a significant parameter for detecting features that would affect fluid flow in pipes.

This first analysis of monocotyledon myosin genes showed that at least five genes, one of which was probably spliced to yield two isoforms, were expressed in maize (Zea mays L.). The complete coding sequence of ZMM1 was determined, as were most of the sequences of two other myosin cDNAs (ZMM2 and ZMM3). ZMM1 and ZMM2 belonged to myosin class XI while ZMM3 was in class VIII. ZMM1 was abundantly expressed in leaves, roots, coleoptiles, and stems. ZMM3 showed a similar distribution but was expressed poorly in pollen. ZMM2 was predominantly expressed in seeds and may be part of a suite of cytoskeletal proteins in reproductive tissues. Phylogenetic analysis suggested that the origin of myosin classes VIII and XI predated that of angiosperms. Immunofluorescence studies using M11L1, a myosin XI antibody specific for the exposed loop 1 head region of myosin, indicated that myosin XI occurred in the cytoplasm of all root tip cells. The highest concentration of myosin XI was in the differentiating epidermal cells. In dividing cells, myosin XI was present near the cytokinetic apparatus at approximately the same concentration seen in other portions of the cytoplasm. Western blot analysis of subcellular fractions indicated that myosin XI was concentrated in mitochondria and low-density membranes. Cell Motil. Cytoskeleton 48:130 ‐148, 2001. © 2001 Wiley-Liss, Inc.

Leaf internal cuticle has not previously been studied in detail, and yet its existence has profound implications for the path of water movement. The internal cuticle forms a uniform layer on the inner periclinal epidermal walls that border substomatal cavities. This cuticle is continuous with the external cuticle through the stomatal pores. The thickness of the internal cuticle on nonstomatal epidermal cells is approximately one-third that of the external cuticle on the same cells. On both the abaxial and adaxial sides of the leaf the internal cuticle forms irregularly shaped islands bordered by mesophyll cells. The size of the islands coincides with the epidermal area of the substomatal cavity. The internal cuticle remains intact and connected to the external cuticle after incubation in cellulytic enzymes. After treatment with sulfuric acid or chloroform, both cuticles remain intact. The autofluorescence of both cuticles is increased by staining with auramine O. These results indicate that large portions of the leaf epidermis are covered by both an internal and an external cuticle.

A new method for the isolation of total RNA from plant tissues is described. This method is designed to circumvent problems resulting from endogenous materials that typically gel during isolation when previously published methods are used. This method is suitable for the isolation of total RNA from plant tissues that contain large amounts of oligosaccharide compounds, and has been used to produce high-quality total RNA from the inner epidermal tissue of onion bulb scale. Using 10 grams of epidermis, it is possible to isolate 0.6 mg of total RNA. This RNA can be directly used in mRNA purification, in vitro translation, and reverse transcription-polymerase chain reaction (RT-PCR). The procedure is simple and takes less than one day.

Cotton (Gossypium hirsutum cv. MD51) fiber cell walls were analyzed with an atomic force microscope to determine the effect of chemical treatments on cell wall organization and topography. Analysis of fibers in either air or water and without any staining or coating produced high-resolution images of cell wall microstructure which could be used for detailed quantitative analysis. Treatment of fibers with 1% H2O2 had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin-diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the height data, amplification of surface area, and integration of the scan line data. Analysis of the fractal dimension enabled untreated and peroxide-treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas. In water, some alkali-treated fibers had microfibrils that were relatively small in diameter while others appeared to consist of crystalline arrays of smaller fibrils.

Summary An acid phosphatase (APase) from onion bulb ( Allium cepa L. var. Sweet Spanish) has been isolated. The purity of the final protein preparation was tested by SDS-PAGE followed by silver staining, which resolved only one protein band. Native-PAGE of the final preparation resolved only one activity band, and it coincided with a single protein band. The molecular mass of the native form of APase was estimated to be 105 ku (kDa) by gel filtration chromatography and the size of the subunit was determined to be 57 ku by SDS-PAGE, indicating that the native enzyme may be a homodimer. The isolated enzyme had its highest specific activity with O-P-Tyr as substrate, its highest specificity constant (V max /K m ) with ATP, and its lowest K m with PEP. The isolated enzyme dephosphorylated a wide range of molecules including ADP, UTP, ITP, GTP, Glycerol-2-P, and O-P-Ser. The enzyme displayed a pH optimum of 5.7 and was strongly inhibited by MgCl 2 and ZnCl 2 . CaCl 2 showed slight inhibition while LiCl had no effect on the enzyme activity. At pH 7.0 and with ATP as a substrate, F-actin activated the activity of the enzyme.

Metallic biomedical devices with nanometer-sized grains (NGs) provide surfaces that are different from their coarse-grained (CG) (tens of micrometer) counterparts in terms of increased fraction of grain boundaries (NG>50%; CG

The objective of the study is to elucidate the interplay between fibronectin-metal hybrid surfaces and osteoblast function. The practical relevance is that a significant initial step in the process of prosthetic integration within a physiological system is the rapid adsorption of proteins, including fibronectin, on the surface of biomedical device. Here, we compare and contrast the cell–substrate interactions on bare and protein-modified surfaces. The protein adsorption on the surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein-adsorbed surfaces. These results support the hypothesis that protein adsorption on artificial biomedical devices can promote bioactivity and regulate biological functions. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3157–3166, 2012.

All 60 Asteraceae species sampled, i.e., two species of Barnadesioideae, 21 species in six tribes in Cichorioideae, and 37 species in nine tribes of Asteroideae, show specific patterns of autofluorescence in abaxial connective base epidermal cells. Two species of Cichorioideae and 15 species of Asteroideae have autofluorescence in adaxial epidermal cells of the connective base. Several taxonomically useful features of the connective base are identified; they include shapes of autofluorescent cells, patterns of distribution of cells with autofluorescent walls, and patterns of distribution of autofluorescence in walls. Cellular features of many species coincide with recognized generic, tribal, and subtribal classifications. The results extend Cassini's characterization of the article antherifere and indicate that the variable features of the connective bases of stamens of Asteraceae could contribute to phylogenetic understanding.

The rapid adsorption of proteins is the starting and primary biological response that occurs when a biomedical device is implanted in the physiological system. The biological response, however, depends on the surface characteristics of the device. Considering the significant interest in nano-/ultrafine surfaces and nanostructured coatings, we describe here, the interplay between grain structure and protein adsorption (bovine serum albumin: BSA) on osteoblasts functions by comparing nanograined/ultrafine-grained (NG/UFG) and coarse-grained (CG: grain size in the micrometer range) substrates by investigating cell-substrate interactions. The protein adsorption on NG/UFG surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability, whereas the effect was less pronounced on protein adsorbed CG surface. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein adsorbed NG/UFG surface. The functional response followed the sequence: NG/UFG(BSA) > NG/UFG > CG(BSA) > CG. The differences in the cellular response on bare and protein adsorbed NG/UFG and CG surfaces are attributed to cumulative contribution of grain structure and degree of hydrophilicity. The study underscores the potential advantages of protein adsorption on artificial biomedical devices to enhance the bioactivity and regulate biological functions.

The sheepshead minnow, Cyprinodon variegatus is a euryhaline fish that inhabits estuaries and coastal marshes where it encounters a wide range of salinities. Many of these areas also have elevated levels of contaminants, creating the potential for toxic ions to interfere with the uptake of ions for osmoregulation. To determine whether the effect of copper on osmoregulatory activity is dependent on the osmotic conditions that individuals have been living at, fish were acclimated for 14 days to 2.5, 10.5 or 18.5 ppt seawater and then exposed to a fixed free cupric ion level (14.6 μM Cu2+) for 6 h. Plasma Na, plasma Cl, wet/dry weight ratio, transepithelial potential difference (TEPD) and branchial Na(+)/K(+)-ATPase activity were determined before and after copper exposure. We also computed Na and Cl equilibrium potentials. Following the salinity acclimation (in fish not yet exposed to copper), fish from the low salinity group (2.5 ppt) had lower TEPD, lower plasma Na levels and higher branchial Na(+)/K(+)-ATPase activity compared to the fish acclimated to higher salinities. No differences in plasma Cl and wet/dry weight ratio were detected. Copper exposure caused a significant decrease in plasma Na levels and Na(+)/K(+)-ATPase activity and an increase in wet/dry weight ratio, but these changes were limited to the 2.5 ppt salinity group. No significant changes in plasma Cl were detected. Copper treatment resulted in a small decrease in TEPD for all except the lowest salinity acclimation group. A comparison of equilibrium potentials with TEPD showed evidence of active transport of both Na and Cl in 2.5 ppt acclimated fish but not for the 10.5 or the 18.5 ppt acclimated fish. Our results show that effects of copper on osmoregulation are dependent on the fish' past salinity regime, and that these effects tend to be more pronounced for euryhaline fish that have been living under hyposmotic conditions.

Longleaf pine (Pinus palustris Mill.) is the most difficult of the southern pines to regenerate by artificial means. One of the basic concerns in regenerating longleaf pine has been in obtaining, processing, and storing seeds of good quality. High seed quality is essential for successful regeneration of the species by either direct seeding or planting. Recommendations for producing and maintaining seed quality at a level equivalent to seeds of the other southern pines are developed in this article. Southern J. Appl. For. 17(4): 180-187.

Structural, autofluorescent, and mechanical characteristics show that the so-called "collar" of the stamen is an elongated basal portion of the connective in Cirsium horridulum. The connective base/filament boundary is precisely demarcated by epidermal cells with thick, autofluorescent walls. In the region ofthe connective base near the anther lobes, the autofluorescence is less intense and the cells are longer, but wall thickness does not decrease. The autofluorescence can be assayed in fresh, frozen, or rehydrated materials, and is resistant to boiling in water and to acetone. Additional features delineate the connective base from the filament. The abaxial and lateral portions of the connective base surface are relatively flat due to the underlying thick secondary walls. Ultrathin sections taken at various points along the length of the connective base and the filament show that cuticle thickness gradually increases from approximately 30 nm in the connective base to 450 nm in the filament. The connective base/filament junction appears to be a weak mechanical link within the stamen. These data establish several new connective base characteristics in Cirsium and define the connective base/filament boundary.

Touching stimulates thistle (Cirsium horridulum Michx.) staminal filaments to rapidly shorten to approximately 70% of their original length. The filaments subsequently re-elongate and regain contractibility within 10min. This thigmonastic movement can be repeated at least 10 times in detached flowers. Filament length is reduced by bathing in 1 M sorbitol, indicating that length change depends on variation in turgor pressure, as is also indicated by plasmolysis observed in contracted filaments. Contraction also depends upon elastic properties of the cuticle. These properties of the cuticle are resistant to boiling in water, chloroform or acid, and treatment with proteases, protein denaturants, sulfatases, and many cell wall degrading enzymes, but are sensitive to cutinase, β-glucuronidase, and boiling in 1 M NaOH. Analysis of carbohydrates from flowers boiled in 1 N NaOH showed that most galacturonic acid residues were extracted from filaments but not from petals (which are inelastic). The filament galacturonic acid residues may form a part of the cuticular contractile mechanism, and/or they may facilitate the bending of the vascular and cortical cells that occurs during contraction. Our results indicate that the elasticity of the stretched cuticle provides the force for the contraction of the filaments and the build-up of turgor causes re-elongation.

Dramatic changes in the localization of conventional non-muscle myosin characterize early embryogenesis in Drosophila melanogaster. During cellularization, myosin is concentrated around the furrow canals that form the leading margin of the plasma membrane as it plunges inward to package each somatic nucleus into a columnar epithelial cell. During gastrulation, there is specific antimyosin staining at the apical ends of those cells that change shape in regions of invagination. Both of these localizations appear to result from a redistribution of a cortical store of maternal myosin. In the preblastoderm embryo, myosin is localized to the egg cortex, sub-cortical arrays of inclusions, and, diffusely, the yolk-free periplasm. At the syncytial blastoderm stage, myosin is found within cytoskeietal caps associated with the somatic nuclei at the embryonic surface. Following the final syncytial division, these myosin caps give rise to the myosin rings observed during cellularization. These distributions are observed with both whole immune serum and affinity-purified antibodies directed against Drosophila non-muscle myosin heavy chain. They are not detected in embryos stained with antiDrosophila muscle myosin antiserum or with preimmune serum. Although immunolocalization can only suggest possible function, these myosin localizations and the coincident changes in cell morphology are consistent with a key role for non-muscle myosin in powering cellularization and gastrulation during embryogenesis.

Metallic materials with submicron- to nanometer-sized grains provide surfaces that are different from conventional polycrystalline materials because of the large proportion of grain boundaries with high free energy. In the study described here, the combination of cellular and molecular biology, materials science and engineering advances our understanding of cell-substrate interactions, especially the cellular activity between preosteoblasts and nanostructured metallic surfaces. Experiments on the effect of nano-/ultrafine grains have shown that cell attachment, proliferation, viability, morphology and spread are favorably modulated and significantly different from conventional coarse-grained structures. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on nanograined/ultrafine-grained substrate. These observations suggest enhanced cell-substrate interaction and activity. The differences in the cellular response on nanograined/ultrafine-grained and coarse-grained substrates are attributed to grain size and degree of hydrophilicity. The outcomes of the study are expected to reduce challenges to engineer bulk nanostructured materials with specific physical and surface properties for medical devices with improved cellular attachment and response. The data lay the foundation for a new branch of nanostructured materials for biomedical applications.

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.

The cuticle is a complex structure of soluble lipids, lipid polymers and polysaccharides. In addition to its functions to reduce water loss and provide a protective barrier, its mechanical properties may be significant to plant growth and development. We investigated the cuticle of Cirsium horridulum Michx. because of its involvement in the thigmonastic contraction of staminal filaments. The staminal filaments and portions of the style are surrounded by a highly elastic cuticle in contrast to the rigid cuticle of the corolla and leaves. Our aim was to determine if the biochemical composition affected the elasticity of the cuticle. We discovered that the ratio of carbohydrates to lipids is 1:7 in floral parts but 2:1 in leaf cuticle. Esterified cutin components represented about 80% of the cuticle and di-hydroxyhexadecanoic acids were the major monomers of cutin, regardless of origin. The cutin of elastic tissues is characterized by a higher content of tri-hydroxy monomers than the cutin of rigid tissues. The data suggest that hydroxyl groups enhance the hydrophilic character of the cuticle and contribute to cuticular elasticity.

Investigations of the longitudinal distribution of the extensibility of staminal filaments of the common thistle (Cirsium horridulum Michx.) showed that the base of the filaments (attachment to corolla) is almost twice as elastic as the apical portion (next to anthers). Boiling leads to a more uniform distribution of extensibility. Using a stress-strain analyzer we investigated the elastic properties of fresh, water-boiled, partially hydrolyzed (acid-boiled), and dehydrated filaments. Stress-strain curves of sinusoidally stretched sets of filaments revealed complex, non-linear behavior with an average modulus of elasticity of 5 MPa·m−2. The phase angle varied from approximately 18 degrees for 0.01-Hz deformations to 84 degrees at 2 Hz, indicating strong viscoelastic components. The viscoelasticity of the filaments indicates that the cell walls have a high ratio of pectin to cellulose. Boiling does not affect Young's modulus, but dehydration does. The technique of applying sinusoidal loads and the analysis of the stress-strain curves proves useful for the assessment of mechanical properties of cell walls, especially for non-growing or contractile tissues.

In summary, we have used a multidisciplinary approach to the analysis of actomyosin-based motility during Drosophila embryogenesis. We have documented the movements of early embryogenesis with modern, video methods. We have characterized the cytoplasmic myosin polypeptide, made specific polyclonal antisera to the molecule, studied its distribution during early embryogenesis, cloned and partially characterized the gene that encodes it, and have recently completed the nucleotide sequence of a nearly full length cDNA that encodes the entire protein-coding region. We have initiated studies on myosin function in living embryos both by direct microinjection of antibodies and through classical genetics. To better understand how myosin function is regulated, we have begun analysis of its light chains. Finally, to investigate the molecular mechanism by which its function is integrated into a labile cytoskeleton, whose architecture is constantly changing, we have also investigated Drosophila spectrins. Together, these studies are designed to shed light on the dynamics of biologic form at the cellular level, with current focus on such complex processes as cytokinesis and morphogenesis.

Microfilaments 50–70 Å (Å = 0.1 nm) in diameter are commonly found in vascular tissue of elongating roots and stems of many plants. Such microfilaments occur in bundles in peripheral regions of elongating or differentiating vascular cells, and are usually oriented parallel to the longitudinal axis of the cells. Although the longest microfilament bundle we have measured is about 15 μm, we suspect that the bundles are at least as long as the cells that contain them. The bundles are 0.1–0.4 μm in width and are often in apparent contact with organelles, but there is no definite indication of their being anchored to plasmalemma or any organelle. The microfilaments are comparable in size and morphology to actin filaments. The distribution of microfilament bundles in the vascular tissue of various plants and the possible function of the bundles is discussed.

Nematodes in the bases of lateral root primordia of Glycine max (L.) Merr. caused abnormal vascular connection development. All lateral roots with single infections at their points of origin in the primary root-the sample included 28 specimens from 2.0-25.0 cm in length-were triarch. The control lateral root sample contained one triarch and 36 tetrarch roots. Affected lateral roots had a peculiar orientation of the xylem poles. Up to 600-700 ,um from the infection site the three xylem poles were oriented at 90Q, 900, and 180? angles. Distally the angle between xylem poles shifted to 1200. All control and infected primary roots were tetrarch. Statistical compariso'i of three parameters, root diameter, vascular cylinder diameter, and primary xylem cross-sectional area showed that only xylem cross-sectional area was significantly different between normal tetrarch and affected triarch lateral roots.

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.

The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.

The distribution of microfilament bundles (MFBs) in the primary tissues ofChamaecyparis obtusa roots has been investigated by electron microscopy. Nomarski differential interference-contrast (NDIC) images of MFBs in sections of embedded materials are also presented to complement the ultrastructural observations. The peripheral phloem parenchyma cells, also known as precursory phloem, generally possess greater numbers of MFBs than do any other cell type. MFBs are apparently absent in the cortical, meristematic or root cap tissues. The number of MFBs seen in a transection of a cell varies according to its position in the ontogenetic sequence. While all the MFBs in peripheral phloem parenchyma cells lie within 2.0 μm from and on occasion contact the plasmamembrane, some MFBs in other phloem and xylem cells are located in the central areas of the cytoplasm. The possible three-dimensional distribution of MFBs in a streaming peripheral phlowm parenchyma cell is discussed.

Cells of conifers contain a variety of intriguing ultrastructural features which are expressed either in specific cell types or at specific stages of cell development. This chapter will describe several such structural features that are found in the primary tissues of roots. Root cells are not yet a common source of explants for cell and tissue culture studies and their potential for clonal propagation remains largely unexplored. However, the data in this chapter are being presented so that, in the future, development in vitro might be compared with cell type-specific structural development which occurs in vivo. In addition, it is hoped that these data will suggest ways in which conifer cells could be used to investigate questions that are of general interest to many cell biologists.

The distribution of F-actin in the complex tissues of a higher plant organ has been visualized by fluorescence labeling the roots of the conifers Chamaecyparis obtusa and Pseudotsuga menziesii with F-actin-specific fluorescent dye-conjugated phallicidin. F-actin is present in the parenchymatous cells of the vascular tissue. Some vascular parenchyma cells possess larger numbers of F-actin-containing structures (microfilament bundles) than are known to exist in any other higher plant cell. Tissue type appears to be an important determinant of the presence or absence of F-actin in a cell. For example, in contrast to vascular cells, cortical cells show no indication of fluorescence labeling of F-actin after incubation with fluorescent phallicidin. Cytoplasmic streaming is seen only in vascular cells and in a pattern that reflects the intracellular distribution of F-actin.