bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping trajectories of pgp-1 membrane protein patches on surfaces of motile fibroblasts reveals a distinct boundary separating capping on the lamella and forward transport on the retracting tail
Authors: Holifield Bruce F. , Jacobson Ken 
Published: April 2021

ABSTRACT
Patches of aggregated membrane proteins on motile fibroblasts are transported from the surfaces of the leading lamella to a site just ahead of the nucleus in the phenomenon known as capping. A major cell surface glycoprotein, Pgp-1 (GP80), was tagged with a monoclonal IgG and then aggregated with fluorescent secondary antibodies. Correlated digitized fluorescence and phase-contrast microscopy were used to map the trajectories of fluorescent Pgp-1 patches located in various regions of the cell surface. The response of patches located in lamellar and nonlamellar regions to spontaneous retraction of the trailing cell margin, or tail was examined in detail. During capping, Pgp-1 patches accumulated at a morphologically distinct site on the cell surface, the ‘null border’, corresponding to the boundary between lamelloplasm and endoplasm and the posterior edge of the dorsal cortical F-actin sheath. Posterior to this site, gradual forward movement of patches accompanied the gradual narrowing phase of the trailing edge retraction that occurs prior to abrupt detachment of the tail, but patches did not actually accumulate at the null border. The rate of forward patch movement was generally greater at positions further behind the boundary. Patch movement correlated approximately with forward organelle movement in the trailing region of the cell. The boundary was also apparent during simultaneous capping and retraction when forward patch transport on the trailing edge and rearward transport of patches across the lamellar surface appeared to converge on the null border. Forward patch transport was strictly confined to regions behind the boundary while retrograde patch transport was confined to the lamellar region ahead of the boundary. Patches are thought to be linked to the cortical cytoskeleton and their transport is discussed in terms of the very different cortical cytoskeletal dynamics occurring in the leading and trailing edges of locomoting cells.
Transcytotic vesicular carriers for polymeric iga receptors accumulate in rat hepatocytes after bile duct ligation
Authors: Larkin Janet M. , Palade George E. 
Published: April 2021

ABSTRACT
In rat hepatocytes, transcytotic vesicular carriers transport the mature 120× 103Mr form of the polymeric IgA receptor (plgA-R), with or without its ligand, plgA, from the sinusoidal to the biliary plasmalemma, where the ectodomain of the receptor is cleaved to produce an 80×103Mr fragment that is secreted into the bile. Here we show that cholestasis induced by bile duct ligation results in the accumulation of transcytotic carriers, identified by the 120 ×103Mr plgA-R and plgA, in the pericanalicular cytoplasm of hepatocytes. To determine the extent of plgA-R accumulation, hepatic total microsomes (TM) were prepared from control and cholestatic rats. Solubilized TM proteins were separated by SDS-PAGE and receptor forms were detected by immunoblotting and autoradiography. Quantitative densitometry of these autoradiograms showed that after duct ligation the 120 × 103Mr receptor accumulated to a level ∼ threefold higher than the control. Concomitantly, immunologically related, novel 124, 90 and 80 × 103Mr proteins (cholestatic antigens) became detectable. Immunoblot analyses of biliary and serum proteins showed that cholestasis resulted in: (1) a marked decrease in the concentrations of the 80 × 103Mr receptor and plgA in the bile, whereas albumin concentrations remained at control levels; and (2) a marked increase in the concentration of the 80 × 103Mr receptor in the serum. Positive sites for plgA-R were localized to the pericanalicular cytoplasm of hepatocytes by indirect immunofluorescence on semithin frozen sections in cholestatic hepatocytes. The sites were more numerous and the positive signal stronger than in controls. One day post-ligation, plgA-positive sites were located to the same pericanalicular cytoplasm of hepatocytes; by three days, however, most plgA appeared in sinusoidal endothelia and Kupffer cells. To validate the vesicular character of the receptor-positive sites, sham-operated and cholestatic livers were processed for either transmission electron microscopy (TEM) or immunogold localization of receptors on thin frozen sections. TEM verified the accumulation of pericanalicular vesicles in cholestatic hepatocytes. Immunogold tests localized plgA-R to pleiomorphic, pericanalicular vesicles, which were increased in number, size and concentration of antigenic sites in cholestatic hepatocytes. These findings indicate that bile duct ligation provides a method for manipulating the in vivo transcytotic pathway and for accumulating previously unstudied transcytotic carriers in hepatocytes.
Rat mast cells degranulate in response to microinjection of guanine nucleotide
Authors: Tatham Peter E. R. , Gomperts Bastien D. 
Published: April 2021

ABSTRACT
Investigation of regulated exocytosis has frequently required the use of permeabilised cell preparations. This has provided evidence that Ca2+-binding and guanine nucleotide-binding proteins can mediate secretion. Since the manner and extent of membrane permeabilisation affect the requirements for Ca2+ and guanine nucleotide, we have introduced such effectors directly into intact, rat peritoneal mast cells by microinjection. During this brief procedure (∼1 s) a glass needle forms a seal with the plasma membrane. Following injection and withdrawal of the needle the membrane reseals without apparent loss of cell contents. Using fluorescent dye, we estimate that the volume injected is ∼5 fl and that the dilution of injected solutes is ∼ 100-fold. Injection of the nucleotides inosine triphosphate, guanosine 5′-[γ-thio]triphosphate (GTPγS) and guanosine 5′-[βγ-imido]triphosphate causes degranulation. The EC50 for GTPγS is ∼10 μM (concentration in the needle) equivalent to an intracellular concentration of ∼100nM. However, the effect of GTPγS is dependent on the presence of Ca2+ in the external medium. This may be explained by a transitory influx of Ca2+ that occurs during impalement, since the seal between needle and membrane will not prevent the movement of small ions. Thus an increase in cytoplasmic Ca2+ appears to be necessary for secretion induced by GTPγS. Using metabolic inhibitors we have investigated the requirement for ATP. Under conditions where [ATP] [has fallen to 60±18μM (S.E.M., n=3) the mast cell agonist, compound 48/80, is unable to induce degranulation, yet injection of GTPγS still activates the cells. Thus the ATP-dependent processes that mediate 48/80-induced secretion are not part of the pathway that is activated by GTPγS and Ca2+.
Decreased expression of fibronectin and the α5β
1 integrin during terminal differentiation of human keratinocytes
Authors: Nicholson Linda J. , Watt Fiona M. 
Published: April 2021

ABSTRACT
We have examined the expression of fibronectin and the α5β1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the α5 and β1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the α5 and β1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the α5β1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.
Attachment strips: A new type of hemidesmosome-like structure in the protozoon Katablepharis ovalis skuja
Authors: Lee Robert Edward , Kugrens Paul 
Published: April 2021

ABSTRACT
Katablepharis ovalis Skuja is a free-living unicellular flagellate with a phagocytic mode of nutrition. Each cell has one or two hemidesmosome-like attachment strips consisting of electron-dense ridges that pass through the plasma membrane to end at the inner wall. Each attachment strip occurs over a group of microtubules of the cytoskeleton and, like the cytoskeleton, is oriented along the long axis of the cell. The attachment strips occur only over the medial area of the cell and are not found over the anterior one-third or posterior one-sixth of the cell. The attachment strips appear to function like hemides-mosomes, attaching the cell to the extracellular matrix. However, the attachment strips in Katablepharis do not have the cytoplasmic component of hemidesmosomes, e.g. fibrils and electron-dense plaques. Among unicellular organisms, hemidesmosomes have been previously reported only in trypanosomes, where hemidesmosomes on an expanded flagellum attach the parasite to the cuticle of the alimentary canal of the insect host The possible relationship between the attachment strips in Katab-lepbaris and the hemidesmosomes in trypanosomes is discussed.
Nuclear distribution of centromeres during the cell cycle of human diploid fibroblasts
Authors: Bartholdi Marty F. 
Published: April 2021

ABSTRACT
The distribution of centromeres in the interphase nuclei of human diploid fibroblasts was analyzed using anti-centromere immunofluorescence and laser scanning confocal microscopy. The positions of the centromeres were placed within the nuclear chromatin distribution and presented some aspects of the dynamics of nuclear structure during the cell cycle. During the Gi phase of the cell cycle many of the centromeres were located in association with nucleoli or fused in chromocenters. A few centromeres were dispersed singly in the euchromatin. During S phase, the fused centromeres dispersed, often forming distinct patterns of rings or lines. At prophase, the centromere immunofluorescence condensed into distinct double dots upon the formation of the prophase chromosomes. Quantitative analysis by both image and flow cytometry showed that the intensity of immunofluorescence started to duplicate in mid S phase, well before the appearance of the double dots. The coalesence of the centromeres during Gi indicated that regions of the chromosome domains remain compacted and possibly sequestered from transcriptional activity. During S phase the chromatin and the coalesced centromeres dispersed for DNA replication. The dynamics of the centromeres and chromatin during the cell cycle seen here are evidence for a higher-order organization of nuclear structure that accompanies DNA transcription and replication.
Genetic analysis of the relationships between the amoebal extranuclear spindle-organizing centre and the plasmodial intranuclear spindleorganizing centre of Physarum during conjugation
Authors: Akhavan-Niaki H. , Mir L. , Oustrin M. L. , Moisand A. , Wright M. 
Published: April 2021

ABSTRACT
Pbysanun amoebae possess an extranuclear spindleorganizing centre (abbreviated SPOC), located in a typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear SPOC without centrioles. In order to ascertain whether, during conjugation, the plasmodial SPOC is derived from the amoebal one or is not related to it, we have constructed amoebal strains possessing two and three SPOCs and we have used as a genetic marker the frequency of polycentric metaphases in order to evaluate the number of SPOCs in the plasmodia. The results of both symmetrical crosses, i.e. between amoebae possessing the same number of SPOCs, and asymmetrical crosses, i.e. between amoebae possessing a different number of SPOCs, show that: (1) the number of SPOCs in plasmodia is dependent upon the number of SPOCs in either one of the two parental amoeba; (2) in no cross does the number of plasmodial SPOCs equal the sum of the parental amoebal SPOCs, but it corresponds to that of only one parent without any polarity of transmission in asymmetrical crosses. These results are consistent with the following model: (1) plasmodial SPOCs are derived from the amoebal ones; and (2) one set of parental SPOCs is lost, destroyed or inactivated in the zygote.
Dynamic regulation of yeast glycolytic oscillations by mitochondrial functions
Authors: Aon M. A. , Cortassa S. , Westerhoff H. V. , Berden J. A. , Spronsen E. Van , Dam K. Van 
Published: April 2021

ABSTRACT
The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investigated in starved yeast cells that were metabolizing glucose semianaerobically. Glycolytic oscillations were triggered after a pulse of glucose by inhibition of mitochondrial respiration with KCN, myxothiazol and antimycin A or in mutants in the bel complex (ubiquinol:cytochrome c reductase) that were largely deficient in respiratory capacity. Inhibition of the adenine nucleotide translocator by preincubation with bongkrekic acid also triggered a train of damped sinusoidal oscillations after glucose addition. The oscillations consisted of cycles of reduction and oxidation of the intracellular pool of nicotinamide nucleotides with periods of 45 s to 1 min and amplitudes of 0.8 mM or lower.
Preincubation with the uncoupler carbonyl cyamide p-(trifluoromethoxy)phenylhydrazone (FCCP) annihilated cyanide-induced oscillations of NAD(P)H. Evidence for de-energization of mitochondrial membranes in vivo was obtained by mitochondrial staining with dimethylaminostyryl-methyl-pyridiniumiodine (DASPMI) of starved cells. The low rates of NADH reoxidation shown by respiratory mutants and the FCCP-treated X2180 strain open up the possibility that mitochondrial dehydrogenases also control glycolytic oscillations. Low rates of cytosolic NADH reoxidation induced by pyrazole, an inhibitor of alcohol dehydrogenase, were also associated with the disappearance of glycolytic oscillations.
From experimental evidence and model calculations we conclude that the modulation of the levels of cytosolic ATP by mitochondrial functions in turn modulates the approach of the dynamic behavior of glycolysis to an oscillatory domain. The mitochondrial NADH dehydrogenase and the glycolytic steps associated with NADH reoxidation downstream from pyruvate appear to provide another control level of glycolysis dynamics in vivo.
Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain
Authors: Hatzfeld Mechthild , Weber Klaus 
Published: April 2021

ABSTRACT
All known intermediate filament (IF) proteins display a consensus sequence at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position −8 was changed to alanine in keratin K8 (K8Y→A-8) and keratin K18 (KlSY→A-8) and leucine at position −4 was changed to glycine in keratin K18 (K18L→G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins.
In standard filament buffer (50 mM Tris-HCl, pH 7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH 7.5) mutant keratins K8Y→A.-8 plus K18Y→A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L→G—4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH 7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.
Hair follicle stem cells? A distinct germinative epidermal cell population is activated in vitro by the presence of hair dermal papilla cells
Authors: Reynolds Amanda J. , Jahoda Colin A. B. 
Published: April 2021

ABSTRACT
Germinative epidermal cells in the lower end bulb region of anagen hair follicles are highly active, and give rise to hair fibres through rapid proliferation and complex differentiation. They have often been termed hair follicle stem cells, but owing to difficulties in isolation and identification their properties have previously only been clearly documented in vivo.
We aimed to isolate and culture germinative cells in vitro, and used microdissection methods to dissect a small but identifiable group of cells from complete follicles. Transmission electron microscopy confirmed that the isolated cells were identical to germinative epidermal cells in situ. SDS-PAGE was used to show that they did not have the same protein composition as epidermis from their immediate proximity (overlying hair matrix), or from other follicular (outer root sheath) and interfollicular (skin basal) regions. Moreover, the germinative cells were found to display morphology and in vitro behaviour that distinguished them from comparative epidermal cells. When cultured in media and on substrata normally conducive to epidermal cell growth they remained in a quiescent state, and did not divide or differentiate. In contrast to other epidermal cells that formed typical pavement-like arrangements, germinative cells remained uniformly small, round and closely packed. However, when cultured in association with hair follicle dermal papilla cells they were radically stimulated into proliferative and aggregative behaviour. Furthermore, they were able to form organotypic-like structures, and exceptionally for skin-derived cell recombinations, a distinct basal lamina at the papilla-germinative cell junction.
These results provide evidence that hair follicle germinative cells have intriguing properties that distinguish them from other follicular epidermis. The finding that they can be activated by dermal papilla cells reflects the intimate nature of the papilla-germinative cell relationship in situ, and should facilitate research into hair growth control mechanisms. The nature of germinative cells is discussed in the wider context of hair follicle stem-cell terminology.
Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes
Authors: Watt Fiona M. , Hudson David L. , Lamb Angela G. , Bolsover Stephen R. , Silver R. Angus , Attchison Michael J. , Whitaker Michael 
Published: April 2021

ABSTRACT
During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thy-midine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+)i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocytes cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and-negative cells were found in the responding and nonresponding populations. Involucrin-positive and-negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serumcontaining medium. Our data therefore indicate that both proliferating (involucrin-negative) and postmitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]1. What role, if any, calcium transients play in triggering keratinocytes DNA synthesis remains to be explored. Nevertheless, our experiments show that the inability of terminally differentiating keratinocytes to divide is not due to the absence of the membrane-[Ca2+]i transduction mechanism.
The nucleolus today
Authors: Hernandez-Verdun Danièle 
Published: April 2021

ABSTRACT
The nucleolus, the most prominent feature of the inter-phase nucleus, is involved in ribosome biogenesis (Warner, 1990). During evolution, the nucleolus first appeared at the time when nuclear envelope compartmentation of the cells was taking place. I would like in this review to provide information showing that the nucleolus is a particular nuclear territory in respect of the compartmentation of nuclear functions.
In eukaryotic cells, the nucleolus is the site at which ribosomal gene transcription takes place and the machinery necessary for the production of the ribosomal subunits is assembled (Hadjiolov, 1985). At this site, the 18 S, 28 S and 5.8 S RNAs are synthesized and assembled with proteins such as ribosomal protein SI (Hügle et al. 1985a) and 5 S RNA.
Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains
Authors: Lichtenstein Anatoly V. , Zaboikin Mikhail M. , Sjakste Nikolai I. , Alechina Raisa P. 
Published: April 2021

ABSTRACT
We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the ‘DNA adherence’ classes DNA-0, DNA-I and DNA-II, respectively. This ‘protein image’ DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA—skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and Si nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.