bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose‐to‐fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and two hyperthermophilic Thermotoga species
Authors: Bandlish Rockey K. , Michael Hess J. , Epting Kevin L. , Vieille Claire , Kelly Robert M.
Published: September 2002

The conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg2+, the temperature optima for the three soluble enzymes were 85°C (SMGI), 95° to 100°C (TNGI), and >100°C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first‐order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg2+. The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60°C and 70°C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg2+ occurred at 70°C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80°C and in the presence of Mg2+, productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose–fructose equilibrium, and enzyme inactivation, was used to examine the potential for high‐fructose corn syrup (HFCS) production at 80°C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co2+ and Mg2+. In the presence of both cations, these enzymes showed the potential to catalyze glucose‐to‐fructose conversion at 80°C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55° to 65°C, but with the additional advantage of higher fructose concentrations. At 90°C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 185–194, 2002.
Optical waveguide lightmode spectroscopy (OWLS) to monitor cell proliferation quantitatively
Authors: Hug T. S. , Prenosil J. E. , Maier P. , Morbidelli M.
Published: September 2002

The use of microscopic observations used for in situ monitoring of cell proliferation in the production of epidermal autografts is not satisfactory. In particular, the identification of the projected cell area from microscopic pictures by image analysis (IA) depends on intensity edges and level of contrasts and is thus limited to subconfluent cultures. Some of these problems can be solved by using optical waveguide lightmode spectroscopy (OWLS), which measures the effective refractive index of a thin layer above an Si(Ti)O2 waveguide surface. In this study the use of OWLS to monitor cell adhesion, spreading, and growth was studied. The sensitivity of the method was investigated by using three different cell lines, two fibroblasts and one hepatoma cell line. Cell proliferation of two strains of fibroblasts and hepatoma cells was monitored up to 2 days with the OWLS. In parallel, cell density was determined at different time points microscopically using an additional window in the measuring chamber. The cell density of fully spread cells (∼4 h after attachment) was found to be proportional to the OWLS signal. In long‐term cultures the influence of the cell density from single cells to confluent cell cultures upon the OWLS signal was investigated. The exponentially growing number of hepatoma resulted in a linear increase of the sensor signal. Due to this and to the fact that the proliferating cells exhibit contact inhibition, it was concluded that the cell contact area must decrease exponentially. The results show the strength of OWLS for monitoring the adhesion and proliferation of anchorage‐dependent cells in applications where an on‐line indicator of the total biomass is needed. Additionally, OWLS provides metabolic information through detection of the cell mass in close contact with the waveguide. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 213–221, 2002.
Macroaffinity ligand‐facilitated three‐phase partitioning (MLFTPP) for purification of xylanase
Authors: Sharma Aparna , Gupta M. N.
Published: September 2002

It is shown that eudragit S‐100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three‐phase partitioning. It was found that 95% eudragit S‐100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t‐butanol to polymer solution at 40°C. Three‐phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t‐butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S‐100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95‐fold purification could be obtained by this process. The purified enzyme showed A single band on SDS‐PAGE. The technique shows promise to develop into a general method that could be termed “macroaffinity ligand‐facilitated three‐phase partitioning (MLFTPP).” © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 228–232, 2002.
The heat shock response of Mycobacterium tuberculosis: linking gene expression, immunology and pathogenesis
Authors: Stewart Graham R. , Wernisch Lorenz , Stabler Richard , Mangan Joseph A. , Hinds Jason , Laing Ken G. , Butcher Philip D. , Young Douglas B.
Published: September 2002

The regulation of heat shock protein (HSP) expression is critically important to pathogens such as Mycobacterium tuberculosis and dysregulation of the heat shock response results in increased immune recognition of the bacterium and reduced survival during chronic infection. In this study we use a whole genome spotted microarray to characterize the heat shock response of M. tuberculosis. We also begin a dissection of this important stress response by generating deletion mutants that lack specific transcriptional regulators and examining their transcriptional profiles under different stresses. Understanding the stimuli and mechanisms that govern heat shock in mycobacteria will allow us to relate observed in vivo expression patterns of HSPs to particular stresses and physiological conditions. The mechanisms controlling HSP expression also make attractive drug targets as part of a strategy designed to enhance immune recognition of the bacterium. Copyright © 2002 John Wiley & Sons, Ltd.
Microarray analysis of bacterial gene expression: towards the regulome
Authors: Kendall Sharon L. , Movahedzadeh Farahnaz , Wietzorrek Andreas , Stoker Neil G.
Published: September 2002

Microarray technology allows co‐regulated genes to be identified. In order to identify genes that are controlled by specific regulators, gene expression can be compared in mutant and wild‐type bacteria. However, there are a number of pitfalls with this approach; in particular, the regulator may not be active under the conditions in which the wild‐type strain is cultured. Once co‐regulated genes have been identified, protein‐binding motifs can be identified. By combining these data with a map of promoters, or operons (the operome), the regulatory networks in the cell (the regulome) can start to be built up. Copyright © 2002 John Wiley & Sons, Ltd.
BµG@Sbase—a microarray database and analysis tool
Authors: Witney Adam A. , Hinds Jason
Published: September 2002

The manufacture and use of a whole‐genome microarray is a complex process and it is essential that all data surrounding the process is stored, is accessible and can be easily associated with the data generated following hybridization and scanning. As part of a program funded by the Wellcome Trust, the Bacterial Microarray Group at St. George's Hospital Medical School (BµG@S) will generate whole‐genome microarrays for 12 bacterial pathogens for use in collaboration with specialist research groups. BµG@S will collaborate with these groups at all levels, including the experimental design, methodology and analysis. In addition, we will provide informatic support in the form of a database system (BµG@Sbase). BµG@Sbase will provide access through a web interface to the microarray design data and will allow individual users to store their data in a searchable, secure manner. Tools developed by BµG@S in collaboration with specific research groups investigating analysis methodology will also be made available to those groups using the arrays and submitting data to BµG@Sbase. Copyright © 2002 John Wiley & Sons, Ltd.
Harvesting the mouse genome
Authors: Botcherby Marc
Published: September 2002

The sequencing of the black 6 mouse (strain C57Bl/6) has reached an important juncture. The BAC fingerprint map is almost complete, the BACs have been end‐sequenced and a seven‐fold coverage whole‐genome shotgun has been assembled. Now the BAC‐by‐BAC sequencing phase is under way and in‐depth comparative analysis can be carried out on regions that have been the subject of targeted sequencing. This paper reviews the progress so far and looks forward to the promises of finished sequence. Copyright © 2002 John Wiley & Sons, Ltd.
Heterologous array analysis in Pinaceae: hybridization of Pinus taeda cDNA arrays with cDNA from needles and embryogenic cultures of P. taeda, P. sylvestris or Picea abies
Authors: van Zyl Leonel , von Arnold Sara , Bozhkov Peter , Chen Yongzhong , Egertsdotter Ulrika , MacKay John , Sederoff Ronald R. , Shen Jing , Zelena Lyubov , Clapham David H.
Published: September 2002

Hybridization of labelled cDNA from various cell types with high‐density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r2 = 0.78 − 0.86), and somewhat lower for embryogenic cultures (r2 = 0.68 − 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r2 = 0.52 − 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue‐specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z‐normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces. Copyright © 2002 John Wiley & Sons, Ltd.
Migration, family structure and children's well‐being: a multi‐level analysis of the second generation of the 1958 Birth Cohort Study
Authors: Verropoulou Georgia , Joshi Heather , Wiggins Richard D.
Published: September 2002

The relationship between moving home, family structure and children's well‐being, is examined in the National Child Development Study (NCDS) Second‐Generation. Well‐being is measured as attainment in maths and reading, and on two behavioural assessments. Multivariate multi‐level modelling allows for heterogeneity both within and between families. We find little to no association between moving home and children's well‐being. Associations between family living arrangements and children's development appear to be mediated by human, financial and social capital, but not, on average, worsened by geographical mobility. Copyright © 2002 John Wiley & Sons, Ltd.
A comparison of Swedish and US fathers' self‐reported use of parental discipline
Authors: Jutengren Göran , Palmérus Kerstin
Published: September 2002

Two samples (pairwise matched in terms of sex of child, age of child, and fathers' education) of fathers from Sweden (n = 50) and the US (n = 50) respectively were interviewed about their use of parental discipline when in conflict with their 38–66‐month‐olds. The results show that, compared with US fathers, Swedish fathers display a range of disciplining approaches from punitive reprimands (i.e., behaviour modification and physical punishment) to restrictive control approaches (i.e., verbal control and physical restraint). No difference was found between Swedish and US fathers in terms of their general tendency to assert compliance when their children misbehave. Copyright © 2002 John Wiley & Sons, Ltd.
Competence in the face of adversity: the influence of early family environment and long‐term consequences
Authors: Schoon Ingrid , Parsons Samantha
Published: September 2002

This paper investigates family characteristics that enable children to achieve competence despite being exposed to socio‐economic adversity, and analyses the long‐term outcomes of this positive adaptation. The main finding is that the protective factors that modify the impact of adversity are context specific, and show age as well as period effects. Positive adaptation during early childhood has long‐term consequences, yet resilient young people cannot completely overcome the experience of early socio‐economic disadvantage. The results indicate that future research must identify specific rather than general protective factors that provide protection against specific risks for young people in specific life contexts. Copyright © 2002 John Wiley & Sons, Ltd.
Amorphin is phosphorylase; Phosphorylase is an alpha‐actinin‐binding protein
Authors: Chowrashi Prokash , Mittal Balraj , Sanger Jean M. , Sanger Joseph W.
Published: September 2002

In a study of myofibrillar proteins, Chowrashi and Pepe [1982: J. Cell Biol. 94:565–573] reported the isolation of a new, 85‐kD Z‐band protein that they named amorphin. We report that partial sequences of purified amorphin protein indicate that amorphin is identical to phosphorylase, an enzyme important in the metabolism of glycogen. Anti‐amorphin antibodies also reacted with purified chicken and rabbit phosphorylase. To explore the basis for phosphorylase's (amorphin's) localization in the Z‐bands of skeletal muscles, we reacted biotinylated alpha‐actinin with purified amorphin and with purified phosphorylase and found that alpha‐actinin bound to each. Radioimmune assays also indicated that phosphorylase (amorphin) bound to alpha‐actinin, and, with lower affinity, to F‐actin. Negative staining of actin filaments demonstrated that alpha‐actinin mediates the binding of phosphorylase to actin filaments. There are several glycolytic enzymes that bind actin (e.g., aldolase, phosphofructokinase, and pyruvate kinase), but phosphorylase is the first one demonstrated to bind alpha‐actinin. Localization of phosphorylase in live cells was assessed by transfecting cultures of quail embryonic myotubes with plasmids expressing phosphorylase fused to Green Fluorescent Protein (GFP). This resulted in targeting of the fusion protein to Z‐bands accompanied by a diffuse pattern in the cytoplasm. Cell Motil. Cytoskeleton 53:125–135, 2002. © 2002 Wiley‐Liss, Inc.
Localised depletion of polymerised actin at the front of Walker carcinosarcoma cells increases the speed of locomotion
Authors: Keller Hansuli , Zadeh Alireza Dehghani , Eggli Peter
Published: September 2002

Spontaneously migrating Walker carcinosarcoma cells usually form lamellipodia at the front. Combined treatment with 10‐5M colchicine and 10‐7M latrunculin A produces large defects in the cortical F‐actin layer at the leading front and suppresses lamellipodia. However, the cortical actin layer at the rear is intact and shows myosin IIA accumulation. These cells, showing no or little detectable cortical F‐actin at the front and no morphologically recognisable protrusions, migrate faster than control cells with lamellipodia and an intact cortical actin layer. This documents that the cortical actin layer or actin‐powered force generation at the front is redundant for locomotion. Colchicine and latrunculin A have synergistic effects in compromising the cortical layer at the front and in increasing the speed of locomotion, but antagonistic effects on the relative amount of F‐actin per cell. Colchicine but not latrunculin A, can increase the proportion of polarised and locomoting cells under appropriate conditions. Locomotion and polarity of cells treated with latrunculin A and colchicine is inhibited at latrunculin A concentrations >10‐7M, by the myosin inhibitor BDM or the ROCK inhibitor Y‐27632. Colchicine and Y‐27632 have antagonistic effects on polarity and the speed of locomoting cells. The data show that locomotion of metazoan cells, which normally form lamellipodia, can be driven by actomyosin contraction behind the front (cell body, uropod). They are best compatible with a cortical contraction/frontal expansion model, but they are not compatible with models implying that actin polymerisation or actomyosin contraction at the front drive locomotion of the cells studied. Cell Motil. Cytoskeleton 53:189–202, 2002. © 2002 Wiley‐Liss, Inc.
Pseudopodium dynamics and rapid cell movement in Dictyostelium Ras pathway mutants
Authors: Chubb Jonathan R. , Wilkins Andrew , Wessels Deborah J. , Soll David R. , Insall Robert H.
Published: September 2002

Loss of either of the Ras pathway members RasS or GefB causes growing Dictyostelium cells to move aberrantly rapidly. In this study, we describe the changes in motility that underlie these phenotypes using computer‐assisted 3D dynamic image analysis. Unexpectedly, the two mutants use different mechanisms to achieve rapid migration. The rasS− cells' motility is characterised by highly dynamic cell morphology, with rapidly extending and retracting pseudopodia. The gefB− cells do not have an unusually dynamic morphology, and achieve their efficient translocation by the continual remodelling of an existing dominant anterior pseudopodium. In spite of these dramatic changes in pseudopodium behaviour, the underlying motility cycle of both mutants remains normal. The levels of F‐actin in both mutant cell lines are significantly elevated with respect to the wild‐type parental cells, suggesting a possible biochemical basis for these emphatic phenotypes. Cell Motil. Cytoskeleton 53:150–162, 2002. © 2002 Wiley‐Liss, Inc.
A talin fragment as an actin trap visualizing actin flow in chemotaxis, endocytosis, and cytokinesis
Authors: Weber Igor , Niewöhner Jens , Du Aiping , Röhrig Ursula , Gerisch Günther
Published: September 2002

A C‐terminal 63‐kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F‐actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re‐programming of cell polarity in response to changing gradients of chemoattractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F‐actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid‐zone of a dividing cell. Cell Motil. Cytoskeleton 53:136–149, 2002. © 2002 Wiley‐Liss, Inc.
Cytoskeletal structure of myoblasts with the mitochondrial DNA 3243A→G mutation and of osteosarcoma cells with respiratory chain deficiency
Authors: Rusanen Harri , Annunen Johanna , Ylä‐Outinen Heli , Laurila Aino , Peltonen Juha , Hassinen Ilmo E. , Majamaa Kari
Published: September 2002

The cytoskeleton, mainly composed of actin filaments, microtubules, and intermediate filaments, is involved in cell proliferation, the maintenance of cell shape, and the formation of cellular junctions. The organization of the intermediate filaments is regulated by phosphorylation and dephosphorylation. We examined cell population growth, apoptotic cell death, and the morphology of cytoskeletal components in myoblast cultures derived from patients with the 3243A→G mutation in mitochondrial DNA (mtDNA) and from control subjects by means of assays detecting cellular nucleic acids, histone‐associated DNA fragments and by immunolabeling of cytoskeletal components. Population growth was slower in the 3243A→G myoblast cultures, with no difference in the amount of apoptotic cell death. The organization of vimentin filaments in myoblasts with 3243A→G was disturbed by randomization of filament direction and length, whereas no disturbances were observed in the other cytoskeletal proteins. Vimentin filaments formed large bundles surrounding the nucleus in mtDNA‐less (ρ0) osteosarcoma cells and in osteosarcoma cells after incubation with sodium azide and nocodazole. We conclude that defects in oxidative phosphorylation lead to selective disruption of the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. Cell Motil. Cytoskeleton 53:231–238, 2002. © 2002 Wiley‐Liss, Inc.
Computer simulation of flagellar movement VIII: Coordination of dynein by local curvature control can generate helical bending waves
Authors: Brokaw Charles J.
Published: September 2002

Computer simulations have been carried out with a model flagellum that can bend in three dimensions. A pattern of dynein activation in which regions of dynein activity propagate along each doublet, with a phase shift of approximately 1/9 wavelength between adjacent doublets, will produce a helical bending wave. This pattern can be termed “doublet metachronism.” The simulations show that doublet metachronism can arise spontaneously in a model axoneme in which activation of dyneins is controlled locally by the curvature of each outer doublet microtubule. In this model, dyneins operate both as sensors of curvature and as motors. Doublet metachronism and the chirality of the resulting helical bending pattern are regulated by the angular difference between the direction of the moment and sliding produced by dyneins on a doublet and the direction of the controlling curvature for that doublet. A flagellum that is generating a helical bending wave experiences twisting moments when it moves against external viscous resistance. At high viscosities, helical bending will be significantly modified by twist unless the twist resistance is greater than previously estimated. Spontaneous doublet metachronism must be modified or overridden in order for a flagellum to generate the planar bending waves that are required for efficient propulsion of spermatozoa. Planar bending can be achieved with the three‐dimensional flagellar model by appropriate specification of the direction of the controlling curvature for each doublet. However, experimental observations indicate that this “hard‐wired” solution is not appropriate for real flagella. Cell Motil. Cytoskeleton 53:103–124, 2002. © 2002 Wiley‐Liss, Inc.
Myosin II dynamics in Dictyostelium: Determinants for filament assembly and translocation to the cell cortex during chemoattractant responses
Authors: Levi Stephanie , Polyakov Mark V. , Egelhoff Thomas T.
Published: September 2002

In the simple amoeba Dictyostelium discoideum, myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G‐protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP‐myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP‐myosin fusions, and with latrunculin A–treated cells, demonstrated that F‐actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A “headless” GFP‐myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor‐based motility may drive translocation. This lack of localization contrasts with previous work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant‐stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant‐regulated myosin II filament assembly and translocation. Cell Motil. Cytoskeleton 53:177–188, 2002. © 2002 Wiley‐Liss, Inc.
Reorganization and translocation of the ectoplasmic cytoskeleton in the leech zygote by condensation of cytasters and interactions of dynamic microtubules and actin filaments
Authors: Fernández Juan , Cantillana Viviana , Ubilla Andrea
Published: September 2002

The formation and bipolar translocation of an ectoplasmic cytoskeleton of rings and meridional bands was studied in interphase zygotes of the glossiphoniid leech Theromyzon trizonare. Zygotes consisted of a peripheral organelle‐rich ectoplasm and an internal yolk‐rich endoplasm. After microinjection of labeled tubulin and/or actin, zygotes were examined by time‐lapse video imaging, immunofluorescence and confocal microscopy. The rings and meridional bands were formed by condensation of a network of moving cytasters that represented ectoplasmic secondary centers of microtubule and actin filament nucleation. In some cases the network of cytasters persisted between the rings. The cytoskeleton had an outer actin layer and an inner microtubule layer that merged at the irregularly‐shaped boundary zone. Bipolar translocation of the rings, meridional bands, or the network of cytasters led to accumulation of the cytoskeleton at both zygote poles. Translocation of the cytoskeleton was slowed or arrested by microinjected taxol or phalloidin, in a dose‐dependent fashion. Results of drug treatment probably indicate differences in the degree and speed at which the cytoskeleton becomes stabilized. Moreover, drugs that selectively stabilized either microtubules or actin filaments stabilized and impaired movement of the entire cytoskeleton. Microtubule poisons and latrunculin‐B failed to disrupt the cytoskeleton. It is concluded that the microtubule and actin cytoskeletons are dynamic, presumably cross‐linked and resistant to depolymerizing drugs. They probably move along each other by a sliding mechanism that depends on the instability of microtubules and actin filaments. Cell Motil. Cytoskeleton 53:214–230, 2002. © 2002 Wiley‐Liss, Inc.
Embryonic wound healing by apical contraction and ingression in Xenopus laevis
Authors: Davidson Lance A. , Ezin Akouavi M. , Keller Ray
Published: September 2002

We have characterized excisional wounds in the animal cap of early embryos of the frog Xenopus laevis and found that these wounds close accompanied by three distinct processes: (1) the assembly of an actin purse‐string in the epithelial cells at the wound margin, (2) contraction and ingression of exposed deep cells, and (3) protrusive activity of epithelial cells at the margin. Microsurgical manipulation allowing fine control over the area and depth of the wound combined with videomicroscopy and confocal analysis enabled us to describe the kinematics and challenge the mechanics of the closing wound. Full closure typically occurs only when the deep, mesenchymal cell‐layer of the ectoderm is left intact; in contrast, when deep cells are removed along with the superficial, epithelial cell‐layer of the ectoderm, wounds do not close. Actin localizes to the superficial epithelial cell‐layer at the wound margin immediately after wounding and forms a contiguous “purse‐string” in those cells within 15 min. However, manipulation and closure kinematics of shaped wounds and microsurgical cuts made through the purse‐string rule out a major force‐generating role for the purse‐string. Further analysis of the cell behaviors within the wound show that deep, mesenchymal cells contract their apical surfaces and ingress from the exposed surface. High resolution time‐lapse sequences of cells at the leading edge of the wound show that these cells undergo protrusive activity only during the final phases of wound closure as the ectoderm reseals. We propose that assembly of the actin purse‐string works to organize and maintain the epithelial sheet at the wound margin, that contraction and ingression of deep cells pulls the wound margins together, and that protrusive activity of epithelial cells at the wound margin reseals the ectoderm and re‐establishes tissue integrity during wound healing in the Xenopus embryonic ectoderm. Cell Motil. Cytoskeleton 53:163–176, 2002. © 2002 Wiley‐Liss, Inc.
Large‐scale morphological survey of mouse retinal ganglion cells
Authors: Sun Wenzhi , Li Ning , He Shigang
Published: September 2002

Five hundred twenty ganglion cells in an isolated whole‐mount preparation of the mouse retina were labeled using the “DiOlistic” method (Gan et al. [2000] Neuron 27:219–225) and were classified according to their morphological properties. Tungsten particles coated with a lipophilic dye (DiI) were propelled into the whole‐mount retina using a gene gun. When a dye‐coated particle contacted the cell membrane, the entire cell was labeled. The ganglion cells were classified into four groups based on their soma size, dendritic field size, and pattern and level of stratification. Broadly monostratified cells were classified into three groups: RGA cells (large soma, large dendritic field), RGB cells (small to medium‐sized soma, small to medium‐sized dendritic field), and RGC cells (small to medium‐sized size soma, medium‐sized to large dendritic field). Bistratified cells were classified as RGD. This study represents the most complete morphological classification of mouse retinal ganglion cells available to date and provides a foundation for further understanding of the correlation of physiology and morphology and ganglion cell function with genetically manipulated animals. J. Comp. Neurol. 451:115–126, 2002. © 2002 Wiley‐Liss, Inc.
Characterization of the optic nerve and retinal ganglion cell layer in the dysmyelinated adult Long Evans Shaker rat: Evidence for axonal sprouting
Authors: Phokeo Vinay , Kwiecien Jacek M. , Ball Alexander K.
Published: September 2002

Myelin in the central nervous system (CNS) is hypothesized to help guide the growth of developing axons by inhibiting sprouting of aberrant neurites. Previous studies using animal models lacking CNS myelin have reported that increasing capacity for sprouting axons is negatively correlated with the degree of myelination. In the present study, we investigated the optic nerves of the recently identified Long Evans Shaker (LES) rat with prolonged dysmyelination of adult axons to determine whether the lack of myelin basic protein (MBP) in adult LES rats could manifest as increases in the population of CNS axons. We observed numerous small, unmyelinated axon profiles (<0.3 μm in diameter) clustered in bundles alongside normal caliber axons in dysmyelinated LES rats but not in normal myelinated Long Evans (LE) rats. These putative axon profiles resembled sprouting axons previously described in the CNS. Moreover, the high number of small putative axon profiles could not be accounted for by any significant increases in the number of ganglion cells and displaced amacrine cells in the ganglion cell layer when compared with normal rats as evaluated by using a variety of techniques. This finding suggests that the observed clusters of putative axon profiles were not due to developmental abnormalities in the retina but to the lack of myelin in the optic nerves of LES rats. The adult LES rat, therefore, may serve as a useful model to study the role of myelin in regulating axon development or axon regeneration after CNS injury in the adult mammalian system. J. Comp. Neurol. 451:213–224, 2002. © 2002 Wiley‐Liss, Inc.
Mitotic activation of proliferative cells in the inner nuclear layer of the mature fish retina: Regulatory signals and molecular markers
Authors: Faillace Maria Paula , Julian David , Korenbrot Juan I.
Published: September 2002

New neurons continuously differentiate within the otherwise mature retina of teleost fish, both under normal conditions and in response to injury. We investigated the effects of surgical injury and intraocular injection of neurotrophic factors on the mitotic rate of proliferative inner nuclear layer cells (PINC). PINC are continually born in the inner nuclear layer and then migrate to the outer nuclear layer (ONL). Surgical excision of a part of a retina activates PINC mitotic activity near and far from the lesion. In the injured eye, up‐regulation of PINC cells is largest in the dorsonasal sector of the retina, regardless of the site of lesion. Up‐regulation extends even to the unlesioned contralateral eye, where it occurs in the same dorsonasal sector. Intraocular injection of ciliary neurotrophic factor mimics the effect of injury on PINC in the treated eye but not on the untreated contralateral retina. We searched for the expression in PINC of Pax6, a transcription factor linked to retinal progenitor cells and found that less than 0.5% of all PINC cells express it. Importantly, the number of Pax6‐expressing PINC does not change significantly in the retinas subjected to any of the experimental manipulations tested. Under normal conditions, the default fate of PINC cells is to migrate to the ONL and, likely, replenish the rod progenitor pool. PINC respond to injury (both surgical and light‐dependent) by increasing their mitotic rate; this increase is long lived, but there are no changes in the expression level of Pax6. PINC probably are a heterogenous cell population that can be specified for ultimate, different purposes: creating rod precursors, creating founder cells, creating cone precursors. Several fates are recognized now, but others may also be possible. J. Comp. Neurol. 451:127–141, 2002. © 2002 Wiley‐Liss, Inc.
Some observations on cortical inputs to the macaque monkey amygdala: An anterograde tracing study
Authors: Stefanacci Lisa , Amaral David G.
Published: September 2002

We have previously described the origins of neocortical inputs to the lateral nucleus of the macaque monkey amygdala based on retrograde tracing studies. Here we report results from studies that have attempted to confirm the projections from several candidate afferent regions using3H‐amino acid autoradiography as an anterograde tracer. We have charted, based on the results of 33 separate injections, the topographic distribution of cortical projections throughout the amygdala. Areas TE and TEO of the inferotemporal cortex, portions of the superior temporal gyrus, and the granular region of the insula project primarily to the lateral nucleus, with little or no innervation of other amygdaloid nuclei. In contrast, orbitofrontal, medial prefrontal, and anterior cingulate regions project primarily to the basal and accessory basal nuclei and provide little innervation to the lateral nucleus. The orbitofrontal and medial prefrontal cortices, but not the anterior cingulate cortex, project to medially situated amygdaloid areas such as the cortical and medial nuclei and to the periamygdaloid cortex. The agranular and dysgranular insula, the parainsula, and rostral portions of the superior temporal gyrus project both to the lateral, basal, and accessory basal nuclei and to the medially situated nuclei. Projections to the central nucleus are particularly prominent from these regions. These data are discussed in relation to the hierarchical processing of sensory information that occurs within the amygdaloid complex. J. Comp. Neurol. 451:301–323, 2002. © 2002 Wiley‐Liss, Inc.
In Patas monkey, glutamic acid decarboxylase‐67 and reelin mRNA coexpression varies in a manner dependent on layers and cortical areas
Authors: Rodriguez Miguel A. , Caruncho Hector J. , Costa Erminio , Pesold Christine , Liu Wen‐Sheng , Guidotti Alessandro
Published: September 2002

In nonhuman and human primates, reelin immunoreactivity is expressed consistently in γ‐aminobutyric acid (GABA)‐ergic interneurons of the three upper cortical layers (Impagnatiello et al. [1998] Proc. Natl. Acad. Sci. U S A 95:15718–15723; Rodriguez et al. [2000] Proc. Natl. Acad. Sci. U S A 97:3550–3555). To understand in detail the pattern of reelin synthesis in GABAergic interneurons of primate neocortex, a quantitative analysis of reelin and of glutamic acid decarboxylase‐67 (GAD67) mRNA‐positive neurons as well as a quantitative analysis of total neuronal density measured by neuron‐specific nuclear protein (NeuN) immunoreactivity was carried out in Patas monkey neocortex (Brodmann's areas 2, 3, 4, 6, 9, 17, 18, and 24). Reelin mRNA is expressed in every cortical area and layer studied, but layer II of each cortical area consistently revealed the largest neuronal population expressing reelin mRNA compared with other layers. The percentages of GAD67‐positive neurons in each layer of the eight cortical areas were 83–98% in layer I, 55–64% in layer II, 37–49% in layer III, 71–89% in layer IV, 54–68% in layer V, and 71–85% in layer VI. The percentages of GABAergic neurons expressing reelin were 86–100% in layer I, 76–84% in layer II, 52–96% in layer III, 23–33% in layer IV, 33–57% in layer V, and 34–54% in layer VI. These findings suggest that there may be two classes of GABAergic neurons that can be differentiated by their ability to express reelin mRNA and reelin protein. This differentiation may have a functional significance, considering that reelin is secreted into the extracellular matrix, where it plays a putative role in the maturation of newly formed dendritic spines and binds selectively to dendritic shafts and to spine postsynaptic densities and presumably to integrin receptors, including α3 subunits (Rodriguez et al. [2000]). J. Comp. Neurol. 451:279–288, 2002. © 2002 Wiley‐Liss, Inc.
Anatomy of the brain neurogenic zones revisited: Fractones and the fibroblast/macrophage network
Authors: Mercier Frederic , Kitasako John T. , Hatton Glenn I.
Published: September 2002

Cytogenesis in adult peripheral organs, and in all organs during development, occurs nearby basal laminae (BL) overlying connective tissue. Paradoxically, cytogenesis in the adult brain occurs primarily in the subependymal layer (SEL), a zone where no particular organization of BL and connective tissue has been described. We have reinvestigated the anatomy of the area considered the most neurogenic in the adult brain, the SEL of the lateral ventricle, in zones adjacent to the caudate putamen, corpus callosum, and lateral septal nucleus. Here, we report structural (confocal microscopy using laminin as a marker) and ultrastructural evidence for highly organized extravascular BL, unique to the SEL. The extravascular BL, termed fractones because of their fractal organization, were regularly arranged along the SEL and consisted of stems terminating in bulbs immediately underneath the ependyma. Fractones contacted local blood vessels by means of their stems. An individual fractone engulfed in its folds numerous processes of astrocytes, ependymocytes, microglial cells, and precursor cell types. The attachment site (base) of stems to blood vessels was extensively folded, overlying large perivascular macrophages that belong to a fibroblast/macrophage network coursing in the perivascular layer and through the meninges. In addition, collagen‐1, which is associated with BL and growth factors during developmental morphogenetic inductions, was immunodetected in the SEL and particularly regionalized within fractones. Because macrophages and fibroblasts produce cytokines and growth factors that may concentrate in and exert their effect from the BL, we suggest that the structure described is implicated in adult neurogenesis, gliogenesis, and angiogenesis. J. Comp. Neurol. 451:170–188, 2002. © 2002 Wiley‐Liss, Inc.
BMP mRNA and protein expression in the developing mouse olfactory system
Authors: Peretto Paolo , Cummings Diana , Modena Chiara , Behrens Maik , Venkatraman Giri , Fasolo Aldo , Margolis Frank L.
Published: September 2002

The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (‐ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6‐ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood. J. Comp. Neurol. 451:267–278, 2002. © 2002 Wiley‐Liss, Inc.
Projections from the amygdaloid complex to the claustrum and the endopiriform nucleus: A Phaseolus vulgaris leucoagglutinin study in the rat
Authors: Majak Katarzyna , Pikkarainen Maria , Kemppainen Samuli , Jolkkonen Esa , Pitkänen Asla
Published: September 2002

The claustrum and the endopiriform nucleus contribute to the spread of epileptiform activity from the amygdala to other brain areas. Data of the distribution of pathways underlying the information flow between these regions are, however, incomplete and controversial. To investigate the projections from the amygdala to the claustrum and the endopiriform nucleus, we injected the anterograde tracer Phaseolus vulgaris leucoagglutinin into various divisions of the amygdaloid complex, including the lateral, basal, accessory basal, central, anterior cortical and posterior cortical nuclei, the periamygdaloid cortex, and the amygdalohippocampal area in the rat. Analysis of immunohistochemically processed sections reveal that the heaviest projections to the claustrum originate in the magnocellular division of the basal nucleus. The projection is moderate in density and mainly terminates in the dorsal aspect of the anterior part of the claustrum. Light projections from the parvicellular and intermediate divisions of the basal nucleus terminate in the same region, whereas light projections from the accessory basal nucleus and the lateral division of the amygdalohippocampal area innervate the caudal part of the claustrum. The most substantial projections from the amygdala to the endopiriform nucleus originate in the lateral division of the amygdalohippocampal area. These projections terminate in the central and caudal parts of the endopiriform nucleus. Lighter projections originate in the anterior and posterior cortical nuclei, the periamygdaloid cortex, the medial division of the amygdalohippocampal area, and the accessory basal nucleus. These data provide an anatomic basis for recent functional studies demonstrating that the claustrum and the endopiriform nucleus are strategically located to synchronize and spread epileptiform activity from the amygdala to the other brain regions. These topographically organized pathways also provide a route by means of which the claustrum and the endopiriform nucleus have access to inputs from the amygdaloid networks that process emotionally significant information. J. Comp. Neurol. 451:236–249, 2002. © 2002 Wiley‐Liss, Inc.
N‐methyl‐D‐aspartate receptor blockade inhibits estrogenic support of dendritic growth in a sexually dimorphic rat spinal nucleus
Authors: Hebbeler Sara Louise , Verhovshek Tom , Sengelaub Dale Robert
Published: September 2002

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons requires the action of both androgens and estrogens. Estrogenic effects are limited to the initial growth of SNB dendrites through 4 weeks of age. During this postnatal period, dendritic growth in other spinal motoneurons is regulated by N‐methyl‐D‐aspartate (NMDA) receptor activation. In this study, we tested whether NMDA receptor activation was involved in SNB dendritic growth and whether the estrogenic support of SNB dendritic growth was dependent on the activation of NMDA receptors. Motoneuron morphology was assessed in normal males, intact males treated daily with the NMDA receptor antagonist MK‐801, castrated males treated with estradiol benzoate (EB), and castrated males treated with both EB and MK‐801. SNB motoneurons were retrogradely labeled with cholera toxin‐horseradish peroxidase at 4 weeks of age (when dendritic length is normally maximal) and reconstructed in three dimensions. Somal area and dendritic length of SNB motoneurons in MK‐801‐treated, intact males were below those of normal males. Dendritic growth was partially supported in EB‐treated castrates, but this growth was blocked by MK‐801 treatment. These results suggest that, as in other motoneurons, dendritic development in the SNB involves NMDA receptors and, furthermore, that the estrogen‐sensitive component of SNB dendritic development requires their activation. J. Comp. Neurol. 451:142–152, 2002. © 2002 Wiley‐Liss, Inc.