bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization
Authors: Cousin B. , Cinti S. , Morroni M. , Raimbault S. , Ricquier D. , Pénicaud L. , Casteilla L.
Published: April 2021

Brown adipocytes are thermogenic cells which play an important role in energy balance. Their thermogenic activity is due to the presence of a mitochondrial uncoupling protein (UCP). Until recently, it was admitted that in rodents brown adipocytes were mainly located in classical brown adipose tissue (BAT). In the present study, we have investigated the presence of UCP protein or mRNA in white adipose tissue (WAT) of rats. Using polymerase chain reaction or Northern blot hybridization, UCP mRNA was detected in mesenteric, epidydimal, retroperitoneal, inguinal and particularly in periovarian adipose depots. The uncoupling protein was detected by Western blotting in mitochondria from periovarian adipose tissue. When rats were submitted to cold or to treatment with a -adrenoceptor agonist, UCP expression was increased in this tissue as in typical brown fat. Moreover, the expression was decreased in obese fa/fa rats compared to lean controls. Morphological studies showed that periovarian adipose tissue of rats kept at 24°C contained cells with numerous typical BAT mitochondria with or without multilocular lipid droplets. Immunocytochemistry confirmed that multilocular cells expressed mitochondrial UCP. Furthermore, the number of brown adipocytes and the density of mitochondrial cristae increased in parallel with exposure to cold. These results demonstrate that adipocytes expressing UCP are present in adipose deposits considered as white fat. They suggest the existence of a continuum in rodents between BAT and WAT, and a great plasticity between adipose tissue phenotypes. The physiological importance of brown adipocytes in WAT and the regulation of UCP expression remain open questions.
Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau
Authors: Takemura Reiko , Okabe Shigeo , Umeyama Takashige , Kanai Yoshimitsu , Cowan Nicholas J. , Hirokawa Nobutaka
Published: April 2021

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2cor tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2cor tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.
Microtubule and F-actin dynamics at the division site in living Tradescantia stamen hair cells
Authors: Cleary Ann L. , Gunning Brian E. S. , Wasteneys Geoffrey O. , Hepler Peter K.
Published: April 2021

We have visualised F-actin and microtubules in living Tradescantia virginiana stamen hair cells by confocal laser scanning microscopy after microinjecting rhodamine-phalloidin or carboxyfluorescein-labelled brain tubulin. We monitored these components of the cytoskeleton as the cells prepared for division at preprophase and progressed through mitosis to cytokinesis. Reorganisation of the interphase cortical cytoskeleton results in preprophase bands of both F-actin and microtubules that coexist in the cell cortex, centred on the site at which the future cell plate will fuse with the parent cell wall. The preprophase band of microtubules is formed from microtubules that polymerise and incorporate tubulin during prophase. The preprophase band of actin may form either by reorganisation of pre-existing filaments or by de novo polymerisation. Both cytoskeletal components disappear from the future division site approximately five minutes prior to the breakdown of the nuclear envelope. Cortical microtubules are undetectable throughout mitosis and cytokinesis, whereas cortical F-actin remains abundant, although it is notably excluded from the division site. The phragmoplast, containing both F-actin and microtubules, expands towards the cortical actin exclusion-zone through a region that has no detectable microtubules or F-actin. The phragmoplast comes to rest in the predefined region of the cortex that is devoid of F-actin. It is proposed that cortical F-actin may act as a “negative” template which could position the phragmoplast and cell plate correctly. This is the first in vivo documentation of F-actin dynamics at the division site in living plant cells.
Nuclear proteins of the bovine esophageal epithelium: II. The NuMA gene gives rise to multiple mRNAs and gene products reactive with monoclonal antibody W1
Authors: Tang Tang K. , Tang Chieh-ju C. , Chen Yin-Ling , Wu Cheng-Wen
Published: April 2021

Treatment of small cells derived from the basal layer of bovine esophageal epithelium, with Triton X-100, urea and sonication resulted in a nuclear residue that was used as an immunogen for generation of monoclonal antibodies directed against nuclear components. One such antibody, designated W1, was found to label the nuclei of all cells examined. In interphase cells, the target antigen of antibody W1 was diffusely distributed in the nucleus. During metaphase, however, the W1 antigen formed prominent crescents at the poles of the mitotic spindle, diminished gradually in anaphase, and finally redistributed into the regenerating daughter nuclei. Western blotting with antibody W1 yielded a prominent polypeptide of Mr ∼230,000. The amino acid sequence, deduced from the nucleotide sequence of several overlapping cDNA clones that span the entire coding region, revealed that the W1 polypeptide was identical to the Nuclear Mitotic Apparatus (NuMA) protein, with a long a-helical central core flanked by two nonhelical domains. Interestingly, most cDNA sequences were identical to each other, except for six sequence blocks which were either inserted or deleted in individual cDNA clones. Analysis of the cDNA sequences of various clones, coupled with polymerase chain reaction amplification of cellular mRNA and genomic Southern blotting with region-specific probes, all indicated that multiple mRNA species were present in U-251 human glioma cells, derived from alternative splicing of the RNA transcript from a single NuMA/W1 gene. Besides the predominant form of the mRNA giving rise to the polypeptide of Mr ∼230,000, two other forms of mRNA, which arise as a result of alternative splicing and which use different translation termination codons, may yield lower molecular weight polypeptide products. Consistent with this notion, polypeptides of Mr ∼195,000 and ∼194,000 have been observed in this and other studies on the NuMA/W1 protein. These data suggest that multiple isoforms of the NuMA polypeptides generated by alternative mRNA splicing may play some important functions which remain to be characterized.
Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes
Authors: Wendell Kim Livezey , Wilson Leslie , Jordan Mary Ann
Published: April 2021

Previous work from this laboratory has indicated that very low concentrations of vinblastine block HeLa cells at mitosis in the presence of a full complement of microtubules and without major disruption of spindle organization. In the present study we analyzed the structural organization of mitotic spindle microtubules, chromosomes and centrosomes by electron microscopy after incubating HeLa cells for one cell cycle with 2 nM vinblastine. We found that mitotic block of HeLa cells by vinblastine was associated with alterations of the fine structure of the spindle that were subtle but profound in their apparent consequences. The cell cycle was blocked in a stage that resembled prometaphase or metaphase; chromosomes had not undergone anaphase segregation. Neither the structure of the microtubules nor the structure of the kinetochores was detectably altered by the drug. However, the number of microtubules attached to kinetochores was decreased significantly. In addition, the centrosomes were altered; the normal close association of mother and daughter centriole was lost, numerous membranous vesicles were found in the centrosomal region, and many centrioles exhibited abnormal ultrastructure and had microtubules coursing through their interiors. These findings are consistent with our previous results and indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole microtubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine.
Induction of tenascin in cancer cells by interactions with embryonic mesenchyme mediated by a diffusible factor
Authors: Hiraiwa Noriko , Kida Hirokatsu , Sakakura Teruyo , Kusakabe Moriaki
Published: April 2021

Human cancer cell lines A431 and MCF7, which do not produce tenascin (TN) in vitro, were found to produce TN when injected into nude mice or co-cultured with the embryonic mesenchyme. The TN expression in the developing A431 solid tumor was demonstrated by immunohistochemistory and by in situ hybridization. Human TN was detected in culture media by western blot analysis using human specific monoclonal antibody (RCB-1). During tumorigenesis, in the early stage, mouse TN was actively induced and deposited in the peri- and intertumor spaces surrounding the developing tumor. Two days later, TN derived from human epithe- lial cancer cells was induced and mainly deposited in the intertumor basement membrane. After this stage, tumor cells were actively producing TN. On the other hand, TN induction in non TN-producing cells, such as A431 and MCF7 cell lines, was also observed in vitro. Although cell lines such as NIH-3T3, φ2 , STO, 2H6, 3E5 and CMT315, had no effect on the TN induction, primary cultured embryonic mesenchyme effectively stimulated the TN expression in the cancer cell lines. This mesenchymal effect decreased with age and was entirely lost postnatally. Furthermore, conditioned media from these embryonic mesenchymes could repro- duce the same effects on TN induction as observed in the co-culture study.

In conclusion, these findings suggest that TN induc- tion in epithelial cancer cells may depend on interac- tions with the surrounding environment, that these interactions may be mediated by a soluble factor(s) derived from the surrounding mesenchyme and that the TN induction observed in the tumorigenesis may reflect histogenesis during the embryonic period.
Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis
Authors: Bayly Alison C. , French Nicola J. , Dive Caroline , Roberts Ruth A.
Published: April 2021

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of estab- lishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcino- gens. In view of speculation that peroxisome prolifera- tors suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apop- totic cell death.

Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocyto- chemistry. Thus, the FaO cell line maintained charac- teristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible mark- ers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium.

The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chro- matin condensation and DNA fragmentation patterns characteristic of cells undergoing apoptotic death. Inter- estingly, no apoptosis was seen in monolayer cells, sug- gesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo. The FaO cell line may be a useful tool to investigate the hypothesis that the suppression of apoptosis plays a crucial role in peroxisome prolifera- tor-induced hepatocarcinogenesis.
Quantitative analysis of stress fiber orientation during corneal wound contraction
Authors: Petroll W. Matthew , Cavanagh H. Dwight , Barry Patricia , Andrews Peter , Jester James V.
Published: April 2021

Previous studies of actin and actin-binding proteins in corneal myofibroblasts suggest the development of a contractile apparatus composed, in part, of F-actin micro-filament bundles, i.e. stress fibers. To better understand the mechanics of wound contraction and the relationship between microfilament bundles and wound closure, we have analyzed the spatial and temporal organization of stress fibers during the process of corneal wound healing. Rabbit corneas (26 eyes) received 6 mm full-thickness, central incisions and were studied at various times for F-actin organization using en bloc (whole cornea) staining with FITC-phalloidin, as well as conventional histological techniques. 3-D datasets (z-series of 40 en face optical sections, 1 m steps) were collected using the Biorad MRC-600 laser scanning confocal microscope at various regions within the wound. At 7 days, 3-D analysis showed randomly oriented, interconnected F-actin filament bundles (stress fibers). Between 7 and 28 days, stress fibers appeared to organize gradually into planes parallel to the wound surface, with a large population achieving a final orientation nearly parallel to the long axis of the wound. Using Fourier Transform analysis techniques, an orientation index (OI) was calculated to quantitate global fiber orientation at each time point. Analysis of variance demonstrated a significant change (P ≤ 0.001) in overall stress fiber orientation from a random distribution at day 7 to an alignment more parallel to the lateral wound borders at day 28. Overall, these data suggest that stress fibers undergo temporal changes in spatial organization that correlate with wound closure, and that wound closure does not involve the development of previously described contractile or tractional forces aligned directly across the wound.
CD44 expression on murine tissues
Authors: Kennel Stephen J. , Lankford Trish K. , Foote Linda J. , Shinpock Sarah G. , Stringer Claude
Published: April 2021

CD44 is a cell surface glycoprotein found on lymphoid and epithelial cells. Its primary function on lymphocytes and macrophages is to mediate interaction with endothelium, while its function on epithelial cells is not known. The protein has many different forms, generated by alternative mRNA splicing and by post-translational modification, which may mediate different functions. During previous work on murine lung tumor cells, mAb 133-13A was isolated and shown to recognize a surface glycoprotein, P100, of 90-100 103 Mr. Amino acid sequence analysis of purified P100 indicates that it is CD44. Since few data exist to indicate which forms of CD44 are present in different normal tissues, mAb 13313A was used to analyze CD44 expression in mouse tissue. Quantitative data on the distribution of CD44(P100) in mice show that spleen, thymus, liver, intestine, uterus and choroid of the eye are major sites of expression. In addition, epithelia of adrenals, esophagus and trachea are CD44(P100) positive. Previous work on human cell lines has implicated a high molecular mass (130–160 103 Mr) form of the glycoprotein as the form expressed in epithelial cells and carcinomas. Isolation of CD44 proteins from lymphoid tissues in the mouse indicate that, as in human lymphoid tissue, the low molecular mass form (80–90 103 Mr) is predominately expressed. These data show that both small (∼81 103 Mr) and large forms of the glycoprotein are expressed in basal epithelia of esophagus and trachea and in salivary gland, while only the small form is expressed in epithelium of the adrenal cortex and in the murine lung and mammary carcinomas studied. While these data cannot distinguish between specific splice variants, they show that the large forms of CD44 are minor components in normal tissue and seem to be found only in basal epithelium. The CD44 of low Mr found in epithelial tissues is probably associated with lymphoid cell types in the tissues.
Epithelial cells retain junctions during mitosis
Authors: Baker Janice , Garrod David
Published: April 2021

It has long been known that cells show reduced cell-sub-stratum adhesion during mitosis in tissue culture, but it is not generally known whether cell-cell adhesion is also reduced. Epithelial cells, both in culture and in tissues, are linked together by several different types of inter-cellular junctions. Are these junctions disassembled when epithelial cells divide?

Cultured epithelial cells were fluorescently stained for desmosomes, tight junctions and zonulae adherentes, and large numbers of dividing cells examined by light microscopy. The results suggested that all three types of intercellular junctions were retained throughout cell division and no evidence for internalization of junctions was obtained. The persistence of intercellular junctions by cultured cells during division was confirmed by electron microscopy. In order to determine whether intercellular junctions were similarly retained by dividing cells in tissues, human colonic mucosal crypt cells and basal keratinocytes were studied by electron microscopy. Both cell types retained intercellular junctions during division. Dividing basal keratinocytes also possessed hemidesmosomal contact with the basement membrane. It is suggested that retention of cellular junctions during division is important for maintenance of tissue integrity and organization.
Receptor-mediated endocytosis of transferrin at the blood-brain barrier
Authors: Roberts Richard L. , Fine Richard E. , Sandra Alexander
Published: April 2021

Rat brains were perfused with a transferrin-peroxidase conjugate (Tf-HRP) to characterize morphologically the endocytic pathway of transferrin in blood-brain barrier endothelial cells. Electron microscopic evaluation of rat brains perfused with Tf-HRP at 4°C and subsequently warmed to 37°C for brief periods of time (2 minutes) showed sequestration of Tf-HRP in clathrin coated pits and vesicles on the luminal membrane of the brain endothelium. After 5 minutes of warming, diaminoben-zidine (DAB) reaction product was present in vesicular structures 250μ500 nm in diameter and in associated tubules morphologically identified as large or sorting endosomes. Recycling endosomes were also heavily labelled at this time point. Almost no DAB reaction product remained in the cerebral endothelial cells when the warming period was longer than 15 minutes. Other rat brains were perfused with Tf-HRP at 30°C for 15 minutes prior to fixation and DAB cytochemistry. In these studies, brain endothelial cells contained large amounts of DAB reaction product, mostly localized in 50–100 nm vesicles and tubules, often in the Golgi region of the cells. Coated pits and vesicles and large endo-somes were also heavily labelled. Transcytosis of Tf-HRP was not identified in either perfusion protocol. Ultrastructural, indirect immunocytochemical localization of transferrin receptors showed that the transferrin receptor is highly polarized at the blood-brain barrier and is localized only on the apical membrane, in contrast to other polarized epithelial cells, like hepatocytes, in which the receptor is present on the basolateral membrane. The evidence supports an iron transport model in which iron-loaded transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. The iron then dissociates from transferrin in endosomal compartments and is transcytosed by unknown mechanisms, while the transferrin is retroendocytosed.
Morphological and biochemical analyses of contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal and transformed cells
Authors: Tanaka Junya , Watanabe Tatsuo , Nakamura Norimasa , Sobue Kenji
Published: April 2021

The expression and intracellular distribution of four contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal fibroblasts and their transformed counterparts by Rous or avian sarcoma virus were compared. By analyzing the isoformal expression of actin, caldesmon and tropomyosin using two-dimensional gel electrophoresis, only tropomyosin showed significant alteration in its isoformal expression accompanied by transformation. Morphological study revealed that in normal cells, myosin, caldesmon and tropomyosin were distributed periodically along stress fibers, but were excluded from focal adhesions (adhesion plaques), at which stress fibers terminate. By contrast, the contractile proteins were concentrated within the protrusions of the ventral cell surface of transformed cells, which are cell-adhesive structures with high motility (podosomes). Regional analysis indicated that the contractile proteins do not show diffuse distribution within podosomes. Myosin, some caldesmon and tropomyosin in association with F-actin were localized in the region surrounding the core domains of podosomes. A major part of the caldesmon was, however, located in the core domain with short F-actin bundles. In order to compare the stability and the molecular organization of stress fibers with that of the short F-actin bundles within podosomes, the dorsal plasma membranes of the cells were removed by lysis and squirting. Then, the ruptured cells were treated with various buffers containing high salt, ATP or Ca2 /calmodulin. Myosin, caldesmon and tropomyosin were strongly associated with stress fibers of the ruptured normal fibroblasts even in a buffer containing high salt or Ca2 /calmodulin. On the other hand, myosin and tropomyosin within podosomes were easily extracted by lysis and squirting. And, the remaining caldesmon in podosomes was separated from the short F-actin bundles with high salt or Ca2 /calmodulin buffer. The present findings suggest that the high motility of podosomes from transformed cells is based on the actomyosin system, and that the stable adherence of focal adhesions of normal cells is due to a lack of this system. The accumulation of contractile proteins and their dynamic association within podosomes might be the cause of the short half-life of the structures. In relation to its localization in the core domain of podosomes without myosin and tropomyosin, the function of caldesmon has been discussed.
Microtubule organization during the cell cycle of the primitive eukaryote dinoflagellate Crypthecodinium cohnii
Authors: Perret Eric , Davoust Jean , Albert Marie , Besseau Laurence , Soyer-Gobillard Marie-Odile
Published: April 2021

The complete microtubular system of the dinoflagellate Crypthecodinium cohnii Biecheler is described, as seen by confocal laser scanning fluorescence microscopy and labelling with anti-β-tubulin antibody. This technique allowed us to observe the organization of the subcortical and internal cytoskeletons and the mitotic microtubular system, and their changes during the cell cycle. These observations are compared with those made in cryosections by light microscopy and in fast-freeze-fixed, cryosubstituted cells by electron microscopy. We show the organization of the cortical microtubules, and in particular of the thick microtubular bundles arranged as a three-pronged fork from which they seem to emanate. This fork emerges from a peculiar cytoplasmic zone at the pole of the cell and is in contact with the region of the kinetosomes, at the cingulum. During the G1 phase, only a single, radial microtubular bundle (a “desmose”) is observable in the inner part of the cytoplasm. One of its ends is near the flagellar bases and the other end is close to the nucleus in the centrosome region. During the S phase, the flagella drop off, the cell encysts and the kinetosomes duplicate. In mitosis, the cortical microtubules and the intracytoplasmic microtubular bundles do not depolymerize. The microtubular fork, desmose and centrosome double and migrate, while the divided kinetosomes stay in the same place. Later, the centrosomes organize the extranuclear spindle, which is connected to the kinetosome region by the microtubular desmose. The convergent end of the threepronged fork seems to be in contact with the centrosome region. In early and mid-prophase, thick microtubular bundles pass through the nucleus in cytoplasmic channels and converge towards the two poles. Asters were never seen at the spindle poles. The channels and microtubular bundles in the spindle double in number during late prophase and lengthen in early anaphase. The spindle bundles diverge in late anaphase, extend to very near the plasma membrane and depolymerize during telophase. The cleavage furrow in which tubulin and actin are characterized appears in anaphase, formed by invagination of plasma membrane in the kinetosome region. The structure and rearrangements of the Crypthecodinium cohnii microtubular system are compared with those of other dinoflagellates and protists and of higher eukaryotes.
Characterization of a novel 63 kDa membrane protein : Implications for the organization of the ER-to-Golgi pathway
Authors: Schweizer Anja , Ericsson Maria , Bächi Thomas , Griffiths Gareth , Hauri Hans-Peter
Published: April 2021

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15°C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.
Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes
Authors: Douaire M. , Belloir B. , Guillemot J.-C. , Fraslin J.-M. , Langlois P. , Mallard J.
Published: April 2021

Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. β-actin and albumin mRNA, as well as albumin secretion, were also assessed.

Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.
Rapid de novo formation of gap junctions between insect hemocytes in vitro: a freeze-fracture, dye-transfer and patch-clamp study
Authors: Churchill Dennis , Coodin Sheppy , Shivers Richard R. , Caveney Stanley
Published: April 2021

Gap junctions form between insect hemocytes (blood cells) when they encapsulate foreign objects in the hemocoel (body cavity). In this study we show that hemocytes from cockroach ( Periplaneta americana) form gap-junctions rapidly in vitro. Freeze-fracture replicas of hemocyte aggregates fixed 5 minutes after bleeding contain gap-junctional plaques. Dye passage was detected between carboxyfluorescein diacetate-labelled and unlabelled hemocytes within 3 minutes of bleeding, when the cells made contact as they flattened rapidly onto coverslips. When double whole-cell voltage-clamp was used to measure gap-junction formation between cells which were pushed together, electrical coupling was detected within one second of cell-cell contact. To prevent extensive flattening, cells were plated onto lipophorin-coated coverslips. Junctional conductance increased in staircase fashion with steps corresponding to an average single channel conductance of 345 pS. Assuming all channels to have this conductance, the maximal accretion rate of channels to the growing junction was one channel per second. Junctional currents and dye-coupling were detected in the absence of Ca2+, indicating that involvement of Ca2+-dependent adhesion molecules is not a prerequisite for gap-junction formation in hemocytes. Hemocytes from distantly related insects (cockroach and moth) form functional gap junctions with each other, suggesting sequence homology among gap-junction proteins in insects. The function of rapid gap-junction formation between hemocytes during encapsulation and wound healing in vivo are discussed.
The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth
Authors: Morris Robert L. , Hollenbeck Peter J.
Published: April 2021

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

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

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

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

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

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

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

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

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.