bluuebunny/crossref_metadata_2025 huggingface dataset:
Invasion of a basement membrane matrix by chick embryo primitive streak cells in vitro
Authors: Sanders E. J. , Prasad S.
Published: April 2021

At the time of gastrulation in the chick embryo the upper epiblast layer penetrates its own basement membrane at the primitive streak so that its cells may invade the underlying tissue space. In so forming the primary mesoderm, the cells undergo a concomitant epithelial-to-mesenchymal transformation. In this study, epiblast tissue has been explanted onto a basement membrane gel in order to examine its invasive potential. Fully ingressed primary mesoderm cells were able to penetrate the gel as individual cells, during the course of which the texture of the gel was disrupted. By contrast, epiblast tissue taken from the immediate vicinity of the primitive streak penetrated the gel, but only as a coherent tongue of cells and without gel disruption. These tongues of cells did not undergo the epithelial-to-mesenchymal transformation, and consequently spread as a epithelial sheet when replated on glass. Thus, the absence of gel disruption correlated with the failure of transformation, suggesting that these two events may be linked and that they may require in situ cell interactions for their manifestation. Tissue from the lateral epiblast failed to penetrate the gel. Instead, this tissue either spread on the gel surface or rounded up into a hollow sphere with the basal surface of the cells innermost. In the former case, despite the cell spreading, no lamina densa was organized beneath the sheet, but in the latter case polarity reversal occurred with the formation of a new lamina densa at the cell-gel interface. These results demonstrate that epiblast cells from the primitive streak region tend to show some inherent invasiveness, while more lateral cells do not. However, although this in vitro system may model such embryonic invasive potential, it cannot elicit a prompt epithelial-to-mesenchymal transformation.
Colour genes, oncogenes and melanocyte differentiation
Authors: Bennett Dorothy C.
Published: April 2021

Pigmentation has long been a favourite and easily used marker in the genetics of diverse organisms. Mammalian integumental pigments are melanins, synthesized by melanocytes in the epidermis and hair bulbs. Our understanding of mammalian pigmentation genes has been advanced significantly in the last few years (Hearing and Jiménez, 1989), partly by the advent of methods for the culture and immortalization of melanocytes. In cultured melanocytes, homozygous recessive germline mutations can be visibly expressed, complemented or even revert spontaneously, as will be discussed. In the mouse there are over 130 mutations that affect coat colour, mapping to well over 50 loci (Silvers, 1979). For the sake of brevity, most of this commentary will be concerned with the recent molecular characterization of mutations at just four of these loci, two of which encode developmentally controlled, melanocyte-specific products while the other two play a part in melanocyte development.
On the mechanism of induction of dna synthesis in cultured arterial smooth muscle cells by leukotrienes: Possible role of prostaglandin endoperoxide synthase products and platelet-derived growth factor
Authors: Palmberg Lena , Åke Lindgren Jan , Thyberg Johan , Claesson Hans-Erik
Published: April 2021

The induction of DNA replication in rat aortic smooth muscle cells (SMCs) by leukotrienes (LTs) was studied in order to elucidate the mechanisms of action in further detail. The effect of LTB4 was blocked by the prostaglandin (PG) synthesis inhibitor indomethacin and the effects of LTC4 and LTD4 were blocked by the cysteinyl-containing leukotriene receptor antagonists FPL 55712 and ICI 198615. These observations suggest that LTB4 and the cysteinyl-containing leukotrienes act via distinct receptors and point to a role for prostaglandin endoperoxide synthase products in bringing about the effect of LTB4. Radioimmunological determinations and analyses of [3H]arachidonic acid metabolism showed that the SMCs were able to synthesize PGI2 (measured as the stable metabolite 6-keto-PGFlα), PGE2, PGF2α, and 15(S)hydroxy-eico-satetraenoic acid (15(S)HETE). Moreover, picomolar concentrations of arachidonic acid, PGI2, PGE2, PGF2β and 15(S)HETE induced DNA replication in the SMCs under serum-free conditions, whereas linoleic acid, 6-keto-PGFlα and 5(S)HETE were inactive in this respect Analysis of conditioned media for mitogenic activity (with or without antibodies against platelet-derived growth factor, PDGF) and for the presence of material competing with radioiodinated PDGF for binding to specific cell surface receptors indicated that LTB4 stimulated release of PDGF or a PDGF-like molecule from the cells. These findings suggest that the growth-promoting effect of LTB4 is mediated via a prostaglandin endoperoxide synthase product and/or PDGF produced by the cells themselves.
Rotating the plane of imposed vibration can rotate the plane of flagellar beating in sea-urchin sperm without twisting the axoneme
Authors: Shingyoji Chikako , Katada Jun , Takahashi Keiichi , Gibbons I. R.
Published: April 2021

When the head of a sea-urchin sperm is held in the tip of a micropipette and vibrated laterally, the flagellum beats in phase with the imposed vibration. Rotation of the plane of pipette vibration around the head axis induces a corresponding rotation of the plane of beating, in both live and reactivated sperm. Detailed analysis of the waveforms occurring at different stages of this rotation shows that the characteristic asymmetry of the flagellar bending waves rotates along with the plane of beat. The positions of small polystyrene beads attached as markers on the axonemes of demembranated sperm flagella appear unaffected by the rotation of the beat plane and asymmetry. The imposed rotation of the waveform is thus the result of a rotation of the coordinated pattern of sliding among the doublet tubules of the axoneme, and is not accompanied by a twisting of the whole axonemal structure. These data indicate that neither the plane of flagellar beat nor the direction of beat asymmetry is tightly dependent upon a structural or chemical specialization of particular members of the nine outer doublet microtubules, but that both are the result of some regulatory structure that can be forced to rotate relative to the outer structure of the axoneme.
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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis
Authors: Andreassen Paul R. , Palmer Douglas K. , Wener Mark H. , Margolis Robert L.
Published: April 2021

We have discovered a novel mitosis-specific human autoantigen that arises at the centromeres of prophase chromosomes, but ultimately participates in formation of an organelle that bisects the cell at late anaphase and during telophase. The organelle, discernible as a three-dimensional disc by confocal microscopy, encompasses the entire midzone diameter, and its distribution survives disassembly of interpolar microtubules by cold temperature treatment and detergent lysis of cells. Cytokinetic furrow contraction proceeds normally in dihydrocytochalasin B (DCB)-treated cells, and antigen distribution in the furrow is unaltered. In DCB, the furrow retracts in early interphase, coincident with loss of normal membrane association with the disc, resulting in the formation of binucleate cells. The midzone disc in both drug-treated and normal cells is present at the correct time and position to play a central role in cytokinesis. By immunocytochemistry, the disc appears to contain myosin but not actin. The position of the disc and the possible presence of myosin suggest that cytokinesis may involve the interaction of the disc organelle with actin in the cell cortex to produce cleavage in mammalian cells.
The drug probenecid inhibits the vacuolar accumulation of fluorescent anions in onion epidermal cells
Authors: Oparka K. J. , Murant E. A. , Wright K. M. , Prior D. A. M. , Harris N.
Published: April 2021

The drug probenecid has been shown to inhibit the vacuolar accumulation of a range of fluorescent anions with differing pK2 values (Lucifer Yellow CH, Cascade Blue hydrazide, sulphorhodamine G, car-boxyfluorescein, FITC) in onion epidermal cells. In the absence of the drug, uptake occurred into the vacuole and was sensitive to extracellular pH. In the presence of the drug, the dyes accumulated exclusively in the cytoplasm and nucleus. Probenecid also induced vésiculation of the tonoplast and caused the cytoplasm to become polar, both of these structural changes being reversible by removal of the drug. In the case of the permeant FITC molecule, probenecid inhibited net uptake into the epidermal cells, and addition of the drug to cells that had already accumulated dye in the central vacuole resulted in rapid dye leakage across the tonoplast. However, the other more impermeant probes failed to leak to the cytoplasm once they had accumulated in the vacuole, even after prolonged exposures to probenecid. The data are discussed in the light of recent evidence for probenecid-sensitive carriers capable of transporting fluorescent anions across the endosomal membrane of macrophages and in relation to the concept of a detoxification system for the sequestration of xenobiotic anions by plant cells.
Cytokeratins are exposed on the outer surface of established human mammary carcinoma cells
Authors: Godfroid Edmond , Geuskens Maurice , Dupressoir Thierry , Parent Isabelle , Szpirer Claude
Published: April 2021

Normal human mammary epithelial cells and established tumour cells of the same origin express three to eight cytokeratins, which are distributed throughout the cytoplasm in the form of intermediate filaments. The combined use of the iodogen and the two-dimensional gel electrophoresis methods has allowed us to demonstrate the presence of cytokeratins 8, 18 and 19 on the outer surface of established human mammary carcinoma cells, in particular MCF-7 cells, while they were absent from the surface of normal mammary cells in primary culture. By ultrastructural immunocytochemistry, these cytokeratins were localized on blebs formed by the cell surface. Cytokeratins 8,18 and 19 were also detected in the culture medium of mammary carcinoma cells.
Possible roles for TGFβ1 in the gastrulating chick embryo
Authors: Sanders E. J. , Prasad S.
Published: April 2021

We have examined the immunocytochemical distribution of TGFβ1 (transforming growth factor β1) in the gastrulating chick embryo, and have correlated the results with the ability of this factor to promote in vitro changes in the phenotype of mesoderm and epiblast cells. The findings, together with the demonstration that exogenous TGFβ1 is also able to modulate extracellular matrix deposition by these cells in culture, are consistent with a role for this factor in the formation and morphogenesis of the early mesoderm.

Immunofluorescence analysis, using an antibody to the amino-terminal fragment of TGFβ1, indicates that this factor is located in, or between, cells of the medial epiblast, Hensen’s node and primitive streak. At Hensen’s node, cells of the hypoblast were also strongly labelled. Ingressed mesoderm cells, lateral to the streak, show considerably stronger and more diffuse labelling than the overlying epiblast cells. Although the fluorescent labelling appears to be associated with the extracellular matrix surrounding the mesoderm cells, it is not bound to hyaluronic acid, which is the preponderant molecule in the matrix at this time in development. When added exogenously to cultures of mesoderm cells growing with epithelial characteristics on fibronectin, TGFβ1 effects an epithelial-mesenchymal transformation within 24 h. The reverse transformation is effected in mesoderm cells grown on laminin, while the epiblast cell phenotype is not affected by this treatment regardless of the substratum. TGFβ1 is also able to down-regulate the deposition of fibronectin by mesoderm cells grown on fibronectin and of epiblast cells grown on laminin, but up-regulate fibronectin deposition by mesoderm on laminin. Similar substratum-dependent changes are seen in laminin deposition, which is down-regulated in mesoderm on laminin and up-regulated in epiblast on laminin. No effect on laminin deposition is seen in either cell type grown on fibronectin. Expression of the fibronectin receptor is also down-regulated by TGFβ1 in mesoderm cells grown on fibronectin, and this may explain the decreased deposition of fibronectin associated with these cells under these conditions. We suggest that these results are consistent with a reinforcing role for TGFβ1 in the transformation that results in the emergence of mesoderm cells at gastrulation. This factor may also be involved in the maintenance of the fibroblastic phenotype of the mesoderm cells after their ingression, by effects on the expression of receptors for extracellular matrix and on the deposition of matrix by these cells during their early morphogenesis.
Smooth muscle α-actin is a marker for hair follicle dermis in vivo and in vitro
Authors: Jahoda Colin A. B. , Reynolds Amanda J. , Chaponnier Christine , Forester James C. , Gabbiani Giulio
Published: April 2021

We have examined the expression of smooth muscle α-actin in hair follicles in situ, and in hair follicle dermal cells in culture by means of immunohistochemistry. Smooth muscle "-actin was present in the dermal sheath component of rat vibrissa, rat pelage and human follicles. Dermal papilla cells within all types of follicles did not express the antigen. However, in culture a large percentage of both hair dermal papilla and dermal sheath cells were stained by this antibody. The same cells were negative when tested with an antibody to desmin. Overall, explant-derived skin fibroblasts had relatively low numbers of positively marked cells, but those from skin regions of high hair-follicle density displayed more smooth muscle α-actin expression than fibroblasts from areas with fewer follicles. 2-D SDS-PAGE confirmed that, unlike fibroblasts, cultured papillacells contained significant quantities of the α-actin isoform.

The rapid switching on of smooth muscle α-actin expression by dermal papilla cells in early culture, contrasts with the behaviour of smooth muscle cells in vitro, and has implications for control of expression of the antigen in normal adult systems. The very high percentage of positively marked cultured papilla and sheath cells also provides a novel marker of cells from follicle dermis, and reinforces the idea that they represent a specialized cell population, contributing to the heterogeneity of fibroblast cell types in the skin dermis, and possibly acting as a source of myofibroblasts during wound healing.
Conserved structural motifs in cyclins identified by sequence analysis
Authors: Nugent Jonathan H. A. , Alfa Caroline E. , Young Tania , Hyams Jeremy S.
Published: April 2021

Cyclins, as regulatory subunits of the ubiquitous p34cdc2 protein kinase, act as key controlling elements of the eukaryotic cell cycle. We have examined published sequences of A- and B-type cyclins for both amino acid and secondary structure homologies. In particular, we sought regions of homology outside the recognised area of sequence conservation known as the ‘cyclin box’, as well as conserved features predicted to lie at the protein surface. Our analysis demonstrates the existence of a number of islands of homology outside the cyclin box, and indicates candidate residues for phosphorylation.

One of these, a motif containing the amino acids SPXXXE/D is also present in fission yeast pl38UC1, another protein known to interact with p34ccfc2. This motif may define a possible p34cdc2 binding or phosphorylation site. A database search revealed that the CDC25 and SCD25 genes of the budding yeast Saccharomyces cerevisiae also contain some of the newly identified motifs, perhaps indicating a common regulatory or degradation pathway.
Handbuch der Gewebe-uehre des mensches für aebzte und studihende, mit 313 holzschnitten
Authors: Kölliker Von A.
Published: April 2021

“ Medicine has arrived at such a stage that Microscopical Anatomy appears to form its foundation, as much as the anatomy of the organs and systems; and a thorough study of Physiology and Pathological Anatomy is impossible without an exact knowledge of the most minute formal elements. It becomes then the duty of those who cultivate this field of science not only to communicate their observations to their fellow-workers and to those who are more profoundly acquainted with medicine, but to enable all those who are concerned with the study of Man, and especially studentsand practitioners, to profit thereby. The present work seeks to perform this task by giving as condensed as possible a view of the relations of the elementary parts of the body and of the more minute structure of the organs; avoiding all polemical discussions, with the exception of a few of the more important points as yet sub judice, and leaving the history of the science completely in the background; but, on the other hand, entering as fully as possible into those points which bear upon Physiology, Pathological Anatomy, and Comparative Histology.”
On certain Appearances occurring in Dentine, dependent on its Mode of Calcification
Authors: James S. , Salter A.
Published: April 2021

In Kölliker and Siebold’s Zeitschrift for 1850, Czermak published a paper, on some points of the minute anatomy of the teeth, whose importance has not, as it seems to me, been sufficiently appreciated in this country. No abstract or translation of this paper has yet appeared in English, though the interest and value of its contents probably equals any single other that has appeared since the earlier writings of Purkinje and Retzius.
Translations: Remarks on the Structure and function of the Retina
Authors: Müller H.
Published: April 2021

The view respecting the physiological function of the various layers of the retina arrived at by Professor Kölliker, from the investigation of the human eye, and which he has detailed in a previous paper in these Transactions, I have, also, been led to adopt in the most essential points, from continued researches respecting that membrane.
On the construction and use of the microscope
Authors: Hannover Adolphe , Goodsib John
Published: April 2021

Dr. Hannover’s book is short, which is a great recommendation, and it contains many judicious and useful observations, and is evidently the production of an experienced microscopist ; but we really can scarcely find in it much that should entitle it to appear in an English dress, as it is quite evident that the author is totally unacquainted with the present state of the microscope in this country, however familiar he may be with those of foreign construction. He does not mention, and appears to be unacquainted with any English microscope maker except Mr. Pritchard. Were he aware of what they have done, and still are daily doing, towards the perfection of the microscope as an instrument of scientific research, Dr. Hannover would surely have referred to the labours of Ross, Powell, and of Smith and Beck ; men who, with the aid of Mr. Lister, have done more in the improvement of the achromatic microscope than all the continental opticians together, and whose instruments, both for optical perfection and mechanical contrivances are, as we believe, unrivalled by those of any country in the world. But we are indeed surprised that his learned English editor should have been content thus to ignore the merits of his countrymen, and to omit all notice of some of the most important improvements in the microscope and its appliances, which have now been long in daily use. Many other proofs of Dr. Hannover’s want of information on the points to which we have referred might be adduced, but we would merely, in justification of what we have said, remark, that he appears to be quite ignorant that object-glasses may be made to adjust to varying thicknesses of the glass employed to cover an object (pp. 25, 59), although the mode in which this adjustment might be made was described by Mr. Ross as long ago as 1837, and has been universally adopted by English opticians since that time. He makes no mention either of any of the more recent, ingenious, and most valuable modes of illumination, to which, for some time past, so much and such successful attention has been paid. We refer more particularly to such contrivances as Gillett’s condenser, and the parabolic reflectors of Wenham and Shadbolt.
On a Substance Presenting the Chemical Reaction of Cellulose, Found in the Brain and Spinal Cord of Man
Authors: Virchow Rudolph
Published: April 2021

It is well known that Carl Schmidt *was the first to discover in the Ascidians the presence of a principle previously known to exist only in plants, viz. cellulose, and to show that it was a constituent of the animal tissue. The researches of Kölliker and Löwig, † of Schacht, ‡ and of Huxley, § have established this important fact. The occurrence of this substance, however, was limited to a comparatively very low class of the Invertebrata, and the further discovery made by Gottlieb, in Euglena viridis, viz. that this infusorium contains paramylon, a body isomerous with starch, also had reference only to a creature in the lowest class of the animal kingdom. || Nothing of the kind, on the other hand, has been known as existing in the vertebrata, and it is only since the discovery by C. Bernard—that the liver produces sugar—that we have had reason to suppose that substances belonging to the amylum series may also have a representative.
On the Structure and Vital Properties of the Contractile Substance of the Lowest Animals
Authors: Ecker Alex
Published: April 2021

These observations relate principally to the nature of the substance of which the body of the Hydra viridis is composed, but the conclusions are applied also to many other of the lower animal forms. It is stated that the entire body of the Polype consists of a homogeneous, sometimes clear, sometimes granular, soft, extensible, elastic, and contractile substance, which is reticulated with clear spaces, containing a more or less clear fluid. Ecker denies that cellular structure is found in any part of the animal, of which structure Corda and Baumgartner would make it entirely to consist. The three layers which are presented in the body and arms of the Hydra, are in immediate connexion, and are distinguished : 1. The external— by the presence in it of the thread-cells and the greater rarefaction of the tissue. 2. The middle—by the green granules and a less broken-up tissue: and 3. The innermost layer— by the brown excretion-granules, and during digestion by various absorbed matters, oil-drops, &c. From this similarity in the structure of the three layers, it is probable they are all equally contractile ; though it is perhaps the middle layer in which this property resides in greatest activity.
Beitrage Zur Mykologie (Contributions to Mycology)
Authors: Fresenius Dr. George
Published: April 2021

Dr. Fresenius commences his work with an expression of regret that Mycology has up to the present time been so little studied; and attributes the neglect partly to the unsightliness (unscheinbarkeit) of the objects of the study. We think, however, that the charge of unsightliness is one which cannot be maintained against fungi in general. The Agaricus muscarius, with its brilliant crimson pileus and snowy gills, the Agaricus rutilans with a cap of more sober crimson and gills of the richest yellow, the Agaricas psittacinus, or Parroquet Agaric, varied as the plumage of the bird after which it is named ;—these, and many others which might be mentioned, fairly rival flowers in beauty. In fact, a well-arranged group of Agarics, Clavarias, &c., gathered from the first wood into which the botanist may wander, would form as pretty a decoration for a lady’s boudoir as any bouquet from the greenhouse. Those persons therefore who, without troubling themselves with scientific details and distinctions, look only to the external charms of the productions of nature, would find ample employment in searching out the beauties of the Fungi. We find them assuming the forms of clubs, mitres, bowls, cups, stars, and even of birds’-nests, and lanterns;* and their colour is as varied as their shapes, and growing as they principally do, upon the mouldering remains of other organisms, they seem intended, as Mr. Lee has happily expressed it, to deck even decay and ruin with beauty.