bluuebunny/crossref_metadata_2025_split huggingface dataset:
The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth
Authors: Morris Robert L. , Hollenbeck Peter J. 
Published: April 2021

ABSTRACT
Although small molecules such as ATP diffuse freely in the cytosol, many types of cells nonetheless position their mitochondria in regions of intense ATP consumption. We reasoned that in the highly elongated axonal processes of growing neurons in culture, the active growth cone would form a focus of ATP consumption so distant from the cell body as to require the positioning of mitochondria nearby via regulated axonal transport. To test this hypothesis, we quantified the distribution and transport behavior of mitochondria in live, aerobically respiring chick sympathetic neurons. We found that in the distal region of actively growing axons, the distribution of mitochondria was highly skewed toward the growth cone, with a sevenfold higher density in the region immediately adjacent to the growth cone than in the region 100 m away. When axonal outgrowth was blocked by substratum-associated barriers or mild cytochalasin E treatment, the gradient of mitochondrial distribution collapsed as mitochondria exited retrogradely from the distal region, becoming uniformly distributed along the axon within one hour. Analysis of individual mitochondrial behaviors revealed that mito-chondrial movement everywhere was bidirectional but balanced so that net transport was anterograde in growing axons and retrograde in blocked axons. This reversal in net transport derived from two separate modulations of mitochondrial movement. First, moving mitochondria underwent a transition to a persistently stationary state in the region of active growth cones that was reversed when growth cone activity was halted. Second, the fraction of time that mitochondria spent moving anterogradely was sharply reduced in non-growing axons. Together, these could account for the formation of gradients of mitochondria in growing axons and their dissipation when outgrowth was blocked. This regulated transport behavior was not dependent upon the ability of mitochondria to produce ATP. Our data indicate that mitochondria possess distinct motor activities for both directions of movement and that mitochondrial transport in axons is regulated by both recruitment between stationary and moving states, and direct regulation of the anterograde motor.
Stromal fibroblasts synthesize collagenase and stromelysin during long-term tissue remodeling
Authors: Girard Marie T. , Matsubara Masao , Kublin Claire , Tessier Marilyn J. , Cintron Charles , Fini M. Elizabeth 
Published: April 2021

ABSTRACT
The process of connective tissue remodeling is an important mechanism contributing to tissue morphogenesis in development and homeostasis. Although it has long been known that remodeling tissues actively mediate collagenolysis, little is understood about the molecular mechanisms controlling this cell-regulated process. In this study, we examined the biosynthesis of collagenase and the related metalloproteinase, stromelysin, during remodeling of repair tissue deposited after mechanical injury to the rabbit cornea. Neither enzyme was synthesized by uninjured corneas; however, synthesis and secretion was detectable within one day after injury. Collagenase accumulated in its latent form while stromelysin appeared to be partially activated. Enzymes were synthesized by cells having a fibroblast phenotype. These cells were found within the stroma. New synthesis was correlated with accumulation of enzyme-specific mRNA. Highest levels of enzyme synthesis were observed in the repair tissue. However, stromal cells outside of the repairing area also synthesized both enzymes. The level of synthesis decreased in a gradient radiating from the repair tissue. Total synthetic levels in a given area of cornea were dependent on both the number of cells expressing enzyme and the rate of enzyme synthesis. Synthesis of collagenase was detected in repair tissue as long as nine months after injury. Our findings provide direct support for the hypothesis that new collagenase synthesis by cells in repair tissue is the first step in collagen degradation during long-term tissue remodeling.
Calcification of chick vertebral chondrocytes grown in agarose gels: a biochemical and ultrastructural study
Authors: Hunter Graeme K. , Holmyard Douglas P. , Pritzker Kenneth P. H. 
Published: April 2021

ABSTRACT
Chick embryo vertebral chondrocytes (CHECOV cells) grown in agarose gels form spherical colonies containing cells of hypertrophic morphology and a metachro-matically staining matrix. Biochemical analysis of these cultures resulted in the following findings. (i) Calcifica-tion of CHECOV cultures can be induced by addition of Pi (at least 1.9 mM) or β-glycerol phosphate (BGP).(ii) Alkaline phosphatase activity reaches a maximal value at the time when mineral deposition is initiated.(iii)Added BGP is converted to Pi; maximal production of Pi occurs at the time of maximal alkaline phosphatase activity. (iv) BGP-supplemented cultures produce a degree of calcification that corresponds to the amount of BGP conversion to Pi.
It can be concluded that Pi is rate-limiting for the calcification of chondrocyte cultures. BGP promotes calcification of these cultures by acting as a substrate for the alkaline phosphatase-mediated production of inorganic phosphate.
Enrichment and characterization of thymus-repopulating cells in stroma-dependent cultures of rat bone marrow
Authors: Prakapas Zita , Denoyelle Monique , Dargemont Catherine , Kroese Frans G. M. , Thiery Jean Paul , Deugnier Marie-Ange 
Published: April 2021

ABSTRACT
The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on preestablished mouse bone marrow stromal cell layers. Cocultured cells were maintained either under steroid-free conditions (Whit-lock/Witte-type culture) or in the presence of 10 7 M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were tested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whit-lock/Witte-type cultures, but not Dexter-type cultures, gave rise intrathymically to significant numbers of rat donor-type Thy-1.1high CD2 CD5low CD43 cells accounting for 50% to 90% of the organcultured cells at day 14. Repopulation of fetal mouse thymic lobes by rat Thy-1.1high cells could be used as a readout assay for initiation of thymopoiesis from bone marrow precursor cells, since 90% of the cells were CD3 /low and TCR /low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintained, if not amplified during the 3-day culture period, leading to at least a 10-fold enrichment as compared to unfractionated bone marrow. Unlike fresh low-density cells before culture, short-term Whitlock/Witte-type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associated with a 10% lymphoid cell subset which did not express the B-lineage-associated antigens revealed by HIS24 (the rat B220 equivalent) and HIS50 mAbs. We propose that unanchored thymus-repopulating cells enriched in Whitlock/Witte-type cultures may represent lymphoid-restricted, T-cell precursors of the bone marrow capable of emigrating and colonizing the thymus.
Cloning of a Ca2+-ATPase gene of Plasmodium falciparum and comparison with vertebrate Ca2+-ATPases
Authors: Kimura Masatsugu , Yamaguchi Yoshiko , Takada Suehisa , Tanabe Kazuyuki 
Published: April 2021

ABSTRACT
A Ca2-ATPase gene was cloned from the genomic libraries of Plasmodium falciparum. From the deduced amino acid sequence of the gene, a 139 kDa protein with a total of 1228 amino acids was predicted. Sequence of a partial cDNA clone of the gene identified two introns near the 3-end at the regions identical to the regions assumed for the Ca2-ATPase gene of P. yoelii, a rodent malaria species. As compared with a variety of Ca2+-ATPases, the P. falciparum Ca2+-ATPase had the highest amino acid sequence homology (78%) to the P. yoelii Ca2+-ATPase, moderate homology (45-50%) to vertebrate sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs), and lowest homology (20%) to a plasma membrane Ca2+-ATPase.
The P. falciparum protein conserved sequences and residues that are important for the function and/or structure of the organellar type Ca2+-ATPase, such as high affinity Ca2+-binding sites, fluorescein isothiocyanate (FITC)-binding regions, and the phosphorylation site, but the protein did not contain calmodulin-binding regions that occur in the plasma membrane type Ca2+-ATPase. Thus we concluded the cloned gene was the organellar type Ca2+-ATPase of P. falciparum. In a region between the phosphorylation site and FITC-binding region, the P. falciparum protein was about 200 residues longer than the rabbit SERCA and lacked a sequence that binds to phospholamban, a protein that regulates the activity of the rabbit SERCA.
Comparison of the two malarial Ca2+-ATPases with site-directed mutants of the rabbit SERCA showed perfect conservation of residues, the mutations of which resulted in dysfunction of the enzyme, but low conservation of residues, the mutations of which affected enzyme activity little. Incorporation of the malarial sequences into wheel diagrams for analysis of 10 transmembrane domains of the organellar Ca2+-ATPase showed a cluster of conserved residues on the surface of each-helix. Our comparisons suggest that the malarial Ca2+-ATPases are useful for identifying residues or regions that are important for the function and/or structure of the organellar Ca2+-ATPase.
Dissociation between the calcium-induced and voltage-driven motility in cochlear outer hair cells from the waltzing guinea pig
Authors: Canlon Barbara , Dulon Didier 
Published: April 2021

ABSTRACT
The waltzing guinea pig, possessing an hereditary progressive deafness, shows pathology to the actin-bearing structures within the hair cells of the organ of Corti. In particular, the affected structures include the stereocilia, the cuticular plate and, as shown in the present study, swollen and disorganized subsurface cisternae. To test whether this pathology affected outer hair cell motility, cells were isolated from waltzing guinea pigs and their age-matched controls and were subjected to either electrical or chemical stimulation. Visual detection thresholds and the magnitude of the electrically-induced length changes were equivalent for both groups. However, when intracellular calcium was increased with either the calcium ionophore, ionomycin or Ca2+/ATP (under permeabilized conditions with DMSO), length changes were significantly reduced for the outer hair cells from waltzing guinea pigs compared to the controls. The average percent length increase induced by 10 μM ionomycin for the outer hair cells from control animals was 2.3 ± 1.7 whereas for postnatal day 4 waltzing guinea pigs it was 1.3 ± 1.7. Postnatal day 7 and 10 waltzing guinea pigs responded with significantly smaller percent length changes. The intracellular concentration of ionic calcium increased similarly for both groups after the application of ionomycin as revealed with the indicator fluo-3. In the permeabilized cells in the presence of Ca2+/ATP, control cells responded with a percent length change of 3.5, whereas, age-matched waltzing outer hair cells responded with barely detectable length changes. When the osmolarity of the external medium was reduced, both the control and waltzing outer hair cells responded with a length change that was proportional to the change in osmolarity, indicating the capability of passive length changes.
In summary, the voltage-dependent motility of isolated outer hair cells from the waltzing guinea pigs is intact, whilst the slow calcium-dependent motility is abnormal. As a result of these findings, the outer hair cells from the waltzing guinea pig allow separations between the slow, metabolically dependent length changes induced by chemical stimulation and the fast, metabolically independent length changes induced by electrical stimulation.
Investigation of microtubule assembly and organization accompanying tension-induced neurite initiation
Authors: Zheng Jing , Buxbaum Robert E. , Heidemann Steven R. 
Published: April 2021

ABSTRACT
Pulling on the margin of embryonic chick sensory neurons induces neurite formation de novo. We find that these neurites contain microtubules within minutes after the application of tension and apparently normal microtubule arrays within 10-20 min. We wished to determine whether these microtubules reflected existing microtubules that were reorganized, e.g. pulled into the neurite by the applied forces, or whether they reflected primarily new assembly of tubulin. We investigated tension-induced neurite initiation in the presence of 4 nM vinblastine, a concentration that poisons net microtubule assembly but does not depolymerize extant polymers, thus separating new assembly from movements of existing microtubules. We find that vinblastine seriously compromises the ability of chick sensory neurons to initiate neurites in response to tension. The few poisoned neurites that did form were abnormal in several respects. In contrast to unpoisoned cells, poisoned neurites were prone to stretching and breaking while pulling, as though they lacked normal structural support. Indeed, poisoned neurites possessed only short microtubule fragments. We conclude that the microtubule array seen in tension-induced neurites reflects primarily new microtubule assembly, rather than existing microtubules that were reorganized to invade the neurite. This implies that tension applied to unpoisoned chick sensory neurons rapidly stimulates new microtubule assembly concomitant with neurite initiation. Examination of the tension-induced microtubules shows that both their spatial pattern and their acetylation are similar to that reported for normal growth cone-mediated neurites.
A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein
Authors: Bossie Cynthia A. , Sanders Marilyn M. 
Published: April 2021

ABSTRACT
A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament pro-tein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF pre-dicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yielded a protein with a SDS-PAGE estimated molecular weight of ∼70 kDa. It contains sequence principles characteristic of class V intermedi-ate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may rep-resent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells.
Conditions affecting depolymerization of actin in plant homogenates
Authors: Andersland John M. , Parthasarathy M. V. 
Published: April 2021

ABSTRACT
The successful isolation of actin depends on the ability to control its polymerization state. Conditions that affect the depolymerization of actin in plant homogenates were determined using a qualitative assay for both polymeric and monomeric forms. In this assay, phalloidin was added to homogenates to stabilize actin polymers, which were separated from monomers by gel electrophoresis and detected by immunoblotting. By this criterion, actin in pea and rice root homogenates was initially in fila-mentous form, but depolymerized rapidly at pH 8 or above. If the pH was neutral to slightly acidic, some actin remained in filamentous form. The presence of Mg2+ or EGTA had little effect on the stabilization of filaments compared to the effect of pH. A slightly acidic pH was not by itself able to induce the polymerization of concentrated, purified pea actin, while Mg2+ was sufficient to polymerize the actin at both neutral and alkaline pH. F-actin binding proteins did not appear to prevent the depolymerization of the filaments in alkaline homogenates. Our data suggest that most actin in root cells is filamentous.
The role of Myf-5 in somitogenesis and the development of skeletal muscles in vertebrates
Authors: Arnold Hans-Henning , Braun Thomas 
Published: April 2021

ABSTRACT
Muscle research has become a paradigm for the investigation of developmental processes that trigger determination and differentiation of specialized cell types in vertebrates. The isolation of a family of muscle regulatory genes which includes MyoD, myogenin, Myf-5 and MRF4 (also called herculin and Myf-6) represented a breakthrough in our attempt to identify important genetic components required for myogenesis (for recent reviews see: Emerson, 1990; Olson, 1990; Weintraub et al., 1991). About twenty years ago, the first experimental evidence was provided in muscle cell culture systems that suggested the existence of a “master gene” that controls the myogenic fate of cells (O’Neill and Stockdale, 1974; Holtzer et al., 1975). This hypothesis has been revived recently by the identification of the myogenic regulatory genes. While MyoD was the first gene to be cloned that had the ability to induce the muscle phenotype in cultured fibroblasts (Davis et al., 1987), it is now clear that the other members of this family have similar properties and may serve overlapping biological functions.
Characterization of a putative Drosophila GTP-binding protein
Authors: Fredieu John R. , Mahowald Anthony P. 
Published: April 2021

ABSTRACT
We generated a set of monoclonal antibodies raised against Drosophila antigen eluted from a lectin affinity column. One antibody, mAb 13D5 recognizes an anti-gen found in the most dorsal regions of the ventral mid-line of Drosophila embryos at stages prior to and during axonogenesis. 13D5 recognizes cells dorsal to the ecto-derm in the extended germ band beginning approx-imately 7 hours after fertilization and in the peripheral nervous system (PNS) at about 13 hours. In addition, 24-hour-old cultures of isolated embryonic neuroblasts possess a number of cells that express the 13D5 antigen and are not recognized by horseradish peroxidase anti-sera. These cells extend ramified processes with mult-iple growth cone-like structures or large individual processes with a broad growth cone structure. 13D5 immunoprecipitates a single band with an apparent molecular mass of 58 kDa and isolated a 1.9 kb EcoRI fragment from the Xgt11 expression libraries. In situ hybridization to staged embryos using the digoxygenin-labeled probe reveals a pattern of expression in cells just lateral to the dorsal-most regions (mesectoderm) of the ventral midline in 8-to 9-hour embryos. In situ hybrid-ization to cultured cells derived from whole embryos reveals several cell types with differing morphologies that express transcript recognized by the digoxygenin-labeled probe. These cells may possess either broad processes containing detectable transcript, or long thin processes with no detectable transcript. Northern analy-sis reveals a 2.1 kb RNA transcript detectable in all embryonic stages. Nucleotide sequence obtained from the 1.9 kb insert reveals homology with the GTP-bind-ing regions of two signal recognition particle receptors (SNRP) isolated from canine and human tissues.
Structural reorganization of the rough endoplasmic reticulum without size expansion accounts for dexamethasone-induced secretory activity in AR42J cells
Authors: Rajasekaran Ayyappan K. , Morimoto Takashi , Hanzel David K. , Rodriguez-Boulan Enrique , Kreibich Gert 
Published: April 2021

ABSTRACT
A striking reorganization of the rough endoplasmic reticulum (RER) from a tubulovesicular (TV-RER) to a stacked cisternal (SC-RER) configuration was observed when the secretory activity of AR42J cells, a cell line derived from a rat pancreatic acinar carcinoma, was induced by dexamethasone. Treatment with 10 nM dexamethasone resulted in a 6.6-fold increase in the intracellular and a 4.6-fold increase in the secreted amylase activity, respectively. On the basis of the morphometric analysis of thin-section electron micrographs it has been previously reported that this increase in secretory activity is accompanied by a 2.4-fold or 30-fold increase in the size of the RER. We have developed a new biochemical method to determine the size of the RER by quantifying the membrane-bound ribosomes. Using this procedure we did not detect any change in the size of the RER after induction of an active secretory state in AR42J cells. Electron microscopic observation showed the predominance of SC-RER in dexamethasone-treated cells compared to the abundance of TV-RER in control cells. Laser scanning confocal microscopy showed a patchy distribution of ER staining in dexamethasone-treated cells compared to more basal localization in control cells. On the basis of our observations we conclude that in AR42J cells the increase in secretory activity induced by dexamethasone is accompanied by a reorganization of the RER rather than by an increase in ER surface area, as reported by others. Our results suggest that SC-RER is a biosynthetically more efficient form of the RER, which is found predominantly in actively secreting cells.
Transepithelial calcium transport in the chick chorioallantoic membrane I. Isolation and characterization of chorionic ectoderm cells
Authors: Akins Robert E. , Tuan Rocky S. 
Published: April 2021

ABSTRACT
The chicken eggshell supplies approximately 80% of the calcium found in the hatchling chick. The mobilization of eggshell calcium into the developing embryo involves the transepithelial transport of large amounts of calcium in a development-specific manner. The cells responsible for the transport of eggshell calcium into the embryonic circulation are the ectodermal cells of the chorioallantoic membrane. In this report, we present a method for the isolation and culture of chorioallantoic membrane ectodermal cells, which are amenable to direct experimental manipulation. Cell preparations are characterized with respect to the expression of an ectoderm-specific cell surface marker (transcalcin, a calcium-binding protein), and a specific enzymatic activity (elevated Ca2+-activated ATPase). Functional assessment of in vitro cellular calcium uptake by 45Ca2+ tracer kinetics indicates the persistence of a temperature-sensitive, rapid-influx pathway similar to that observed in vivo. The preparations of primary ectodermal cells present an in vitro system applicable to the experimental analysis of calcium metabolism and transport by the chick chorioallantoic membrane.