bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the expression of interleukin‐8 induced by 25‐hydroxycholesterol in retinal pigment epithelium cells
Authors: Catarino Steve , Bento Carla F. , Brito Ana , Murteira Eliana , Fernandes Alexandre F. , Pereira Paulo
Published: February 2012

Purpose:  This study aimed at elucidating the molecular mechanisms involved in the regulation of IL‐8 production by several oxysterols in retinal pigment epithelium (RPE) cells.

Methods:  A human cell line from RPE (ARPE‐19) was used to test the role of cholesterol and several oxysterols (25‐OH, 7‐KC and 7β‐OH) in the expression and secretion of IL‐8. Expression of IL‐8 was assessed by real‐time PCR, while IL‐8 secretion was evaluated by ELISA. PI3K‐, MEK1/2‐, ERK1/2‐ and NF‐κB‐specific inhibitors were used to assess the specific role of the several players on the regulation of IL‐8 production by oxysterols. A gene‐reporter assay for AP‐1 activity was also conducted to evaluate the putative role of this transcription factor on IL‐8 expression induced by oxysterols.

Results:  Here, we demonstrate that 25‐OH specifically increases transcription and secretion of the cytokine IL‐8 in ARPE‐19 cells. Indeed, treatment of ARPE‐19 with 25‐OH, but not with 7‐KC, 7β‐OH or cholesterol, induced the secretion of IL‐8 from cells. 25‐OH also induced the activation/phosphorylation of ERK1/2 through a mechanism dependent on MEK, ERK1/2 and PI3K kinase activity. Real‐time PCR and ELISA experiments demonstrated that 25‐OH increased transcription and secretion of IL‐8 through a mechanism that is dependent on ERK1/2 and PI3K activity. Furthermore, 25‐OH triggered the activation/phosphorylation of the AP‐1 component c‐Jun and, consistently, increased the transcriptional activity of AP‐1. Additionally, we also found that 25‐OH decreases the levels of IκB and increases the nuclear levels of NF‐κB p65 subunit and that inhibition of NF‐κB activity partially prevents the increased secretion of IL‐8 induced by 25‐OH.

Conclusions:  The results presented in this study suggest a role for 25‐OH in inducing IL‐8 production through pathways that are likely to involve AP‐1 and NF‐κB in ARPE‐19 cells. Our data may also provide new molecular targets for the treatment of AMD.
Density‐dependent inhibition of growth: fractionation of inhibitory diffusible factor(s) released by dense cultures of 3T3 cells
Authors: Harel L. , Blat C. , Chatelain G.
Published: February 2012

Different data have suggested that the density‐dependent inhibition of growth in normal 3T3 cells is due to the release of inhibitory factor(s) into the medium. The present results provide more evidence supporting this hypothesis. From conditioned medium of dense cultures of 3T3 cells, we fractionated a thermolabile inhibitory diffusible factor (I.D.F.) with a molecular weight of about 40.000 This factor decreased the [11C]‐inosine incorporation into purine nucleotides and into nucleic acids of stimulated 3T3 cells. DNA synthesis, determined by [11C]‐thymidine incorporation into nucleic acids, and protein synthesis (determined by the protein content of the cultures maintained in the presence or absence of I.D.F.), were also decreased. The possibility that I.D.F. is involved in density‐dependent inhibition of growth is discussed.
Scanning electron microscope cytochemistry of normal and leukaemic leukocytes
Authors: Soligo D. , Lambertenghi‐Deliliers G. , de Harven E.
Published: February 2012

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.
The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis, storage and release of the secretory products
Authors: Marcinkiewicz M. , Bouchaud C.
Published: February 2012

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40‐45 nm separates the ribosome‐coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17‐21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.
Ultrastructural changes in the pars intermedia of the goldfish kept in calcium‐free environments
Authors: Olivereau M. , Chambolle P. , Dubourg P.
Published: February 2012

The PAS‐positive‐calcium‐sensitive (Ca‐s) cells of the pars intermedia (PI) were studied in goldfish kept in fresh water (FW), deionized water (DW), 1/3 sea water (SW) and 1/3 Ca‐free SW. Ultrastructural studies show that Ca‐s cells of control goldfish kept in FW have a low activity with elongated or deeply indented nuclei. This activity is slightly reduced after 19 days in 1/3 SW. A considerable stimulation of most Ca‐s cells is noted in goldfish kept in DW for 20 or 40 days. The stimulation is similar in 1/3 Ca‐free SW, but it affects sometimes a smaller percentage of cells and may be less marked in peripheral areas of the PI. Exocytotic figures are more numerous in Ca‐s cells of goldfish in 1/3 Ca‐free SW than in DW. A basal lamina is rarely present and direct contacts between PI cells and nervous tissue are frequent, although a single synaptic contact with a type B fiber was observed. MSH cells are not affected in goldfish kept in DW. They are stimulated in 1/3 Ca‐free SW: the physiological significance of this response remains unclear. Few agranular (Agr) cells are scattered in the PI. Evident changes are not observed in the different environments. The present ultrastructural data support the hypothesis that the Ca‐s cells of the PI secrete a factor involved in calcium regulation in some teleosts.
Nuclear matrix, hnRNA, and snRNA in friend erythroleukemia nuclei depleted of chromatin by low ionic strength EDTA
Authors: Long B. H. , Ochs R. L.
Published: February 2012

A novel and rapid procedure is described for the preparation of chromatin‐depleted nuclei (CDN) from Friend erythroleukemia cells under conditions that avoid the use of high salt concentrations. By this procedure isolated nuclei that had previously been incubated with DNase I to partially digest DNA were washed twice in 2 mM EDTA to extract the chromatin. The resulting structures contained 1% of DNA, 65% of total RNA, 60‐80% of hnRNA, 74% of snRNA, 29% of protein, and 2% of histones contained in isolated nuclei. Electron microscopy revealed intact, spherical structures similar in diameter to isolated nuclei and consisting of dense networks of fibrils 50‐100 A thick surrounded by distinct nuclear laminae. Although no morphological evidence was found for residual nucleoli, C23, a nucleolus‐specific phospho‐protein, remained centrally localized in CDN, while Sm antigen specific for snRNPs was diffusely localized but absent from central regions. Addition of 2 mM MgCl2 to CDN resulted in the reformation of morphologically distinguishable residual nucleoli. These studies suggest that nucleolar morphology is, in part, dependent upon divalent cations and components unique to the nucleolar matrix and demonstrate that little randomization of nuclear and nucleolar matrix fibrils occurs during CDN isolation in the absence of divalent cations.
Localization of serotonin and dopamine in the brown adipose tissue of the rat and their variations during cold exposure
Authors: Mory G. , Combes‐George M. , Nechad M.
Published: February 2012

The variations of several biogenic amines in brown adipose tissue (BAT) during cold exposure were studied and their localization investigated with histological methods. The study of serotonin and its metabolite 5‐HIAA suggests that BAT serotonin is mobilized during acute and chronic cold exposure. This amine was found to be principally stored, together with histamine, in mast cells. The mast cell number in BAT was doubled during cold adaptation, as was the histamine content of the tissue. Using radio‐enzymatic assay and high pressure liquid chromatography, only small amounts of dopamine were found in BAT. Since no specific dopamine‐storing structure was detected (for example SIF cells), this low amount of dopamine is probably the precursor pool for noradrenaline synthesis and is most likely stored in the noradrenergic innervation of the tissue. BAT is known to be sensitive to both exogenous serotonin and exogenous dopamine; according to our results serotonin could play a role in BAT regulation while the role of dopamine remains hypothetical.
Development of interscapular brown adipose tissue in the hamster. II ‐ Differentiation of transplants in the anterior chamber of the eye: role of the sympathetic innervation
Authors: Nechad M. , Olson L.
Published: February 2012

The relationship between brown adipose tissue (BAT) and its sympathetic innervation during development was investigated by transplantation of undifferentiated (white fat‐like) hamster BAT into the anterior eye chamber of adult hamsters. Such transplants are known to be revascularized and reinnervated by the vessels and the nerves of the host iris. The morphology of the BAT transplants was analysed during the post‐operative weeks by light and electron microscopy, and the ingrowth of sympathetic nerve fibres from the iris was followed by radioautography. BAT appeared to differentiate in oculo, i.e. presented increasing amounts of adipocytes with multilocular fat deposits and abundant, well‐developed mitochondria, but only after a delay of approx. 10 days, and remained much fatter than in situ. The establishment of the sympathetic innervation was not synchronous with the revascularization process. It occurred simultaneously with the morphological differentiation of the BAT transplants, and the nerve fibre density remained low. In the absence of sympathetic innervation, i.e. when the host irides were sympathectomized prior to transplantation, BAT still differentiated, but the process was further delayed and the proportion of differentiated brown adipocytes after 20 days in oculo was clearly lower than in control transplants. It is concluded that the sympathetic innervation in BAT is involved in the regulation of differentiating activity in the tissue, but is not obligatory for differentiation to occur.
Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms
Authors: Gautron J. , Rieger F. , Blondet B. , Pinçon‐Raymond M.
Published: February 2012

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three‐phase curve in function of time after denervation. There is a rapid initial fall 0‐3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T‐tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.
Morphological study of cell organelles during development. I ‐ The nuclear sac and the endoplasmic reticulum on the rat nephron
Authors: Gaffiero P. , Bergeron M. , Thiery G.
Published: February 2012

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3‐0.6 micron) of kidney tissue were made after a five‐day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80‐100 kV. Studies were performed on rat foetuses from 18‐21 days of gestation, on newborns, and on 2‐20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.
Comparative effect of atropine on the adrenergic and muscarinic stimulation of phospholipid 32P labelling in isolated parotid cells: atropine, a possible blocker of alpha‐adrenergic receptors
Authors: Imhoff V. , Rossignol B.
Published: February 2012

The inhibitory effect of atropine on phospholipid 32P labelling stimulated by muscarinic or alpha‐adrenergic agonists was studied in isolated parotid cells. Atropine (10(‐11) to 10(‐4) M) had no effect on phospholipid 32P labelling in unstimulated cells. In contrast, 10(‐8) to 10(‐7) M atropine provoked a competitive inhibition of the cholinergic stimulation (i.e. this effect was completely wiped out at high agonist concentration). The atropine app. KD for the muscarinic receptor was 5 × 10(‐9) M. Moreover, atropine inhibits the adrenergic stimulation of phospholipid 32P labelling by decreasing the efficacity and potency of the adrenergic agonists. The atropine app. KD for the alpha‐adrenergic receptor can be estimated at 10(‐5) M. This inhibition of alpha‐adrenergic stimulation appears to be specific since atropine was without effect on the substance P or beta‐adrenergic stimulation. At very low concentration (10(‐10) — 10(‐9) M) atropine seems to be a modulator (activator) of the muscarinic or adrenergic agonist‐receptor complex. From the present data, it is suggested that atropine, besides its classical blocker effect at the muscarinic receptor, at high concentration is a specific alpha‐adrenergic antagonist.
Ribosomal DNA sequences detected in malaria parasites by cytochemical hybridization
Authors: Cornelissen A. W. , Langsley G. , Walliker D.
Published: February 2012

A procedure in which fluorochrome‐labelled RNA is hybridized in situ to homologous DNA sequences was used to investigate the possible application of this method to the human malaria parasite Plasmodium falciparum. Rhodamine‐labelled ribosomal RNA stained the nuclei of the parasites after cytochemical hybridization. This result demonstrates that ribosomal RNA genes can be visualised. It was estimated that the hybridization efficiency was greater than 40%.
Effects of phorbol esters on multiplication and differentiation of mammary cells
Authors: Martel P. , Houdebine L. M. , Teyssot B. , Djiane J.
Published: February 2012

Three phorbol esters: phorbol‐12,13‐dibutyrate (PDB), 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) and phorbol‐12,13‐diacetate (PDA) were added at various concentrations (10(1)‐10(4) ng/ml) to the culture medium of rabbit mammary explants. The three phorbol esters inhibited the mitogenic signals of insulin, EGF and prolactin. They also prevented the induction of casein synthesis and casein mRNA accumulation by prolactin. In all cases, the most potent inhibitor was PDB. PDB did not alter the binding of prolactin to its receptors. The three phorbol esters added to mammary cell membranes with prolactin prevented the generation of the hormonal relay which specifically stimulates casein gene transcription when added to isolated nuclei. These data suggest that phorbol esters act directly at the membrane level where they interrupt the transfer of the hormonal messages, through a mechanism which may not be related to their promoter potency.
Megakaryocytes separation in homogeneous classes by unit gravity sedimentation: physico‐chemical, ultrastructural and cytophotometric characterizations
Authors: Dupont H. , Dupont M. A. , Bricaud H. , Boisseau M. R.
Published: February 2012

Separation by velocity sedimentation at unit gravity according to the STAPUT system of Miller and Phillips was applied to a population of rabbit megakaryocytes previously enriched by density gradient centrifugation. By this means, 80,000 to 100,000 megakaryocytes with 100% purity were collected in eight fractions according to size for a sedimentation velocity of 52 to 30 mm/hr. DNA‐Feulgen cytophotometric measurements show significant correlation between megakaryocyte size and ploidy. The study of the eight purified fractions is of particular interest because it reflects megakaryopoiesis evolution. The different stages of megakaryocyte maturation of each fraction were analysed by transmission and scanning electron microscopy and were correlated to ploidy level. Thrombopoietic megakaryocytes with grape‐like appearance were found in ploidy fractions 8n to 128n. Cytophotometric determinations of nucleohistones revealed several populations.
Basement membrane maintenance in cultures of isolated thyroid follicles
Authors: Alquier C. , Athouel‐Haon A. M. , Mazzella E. , Michel‐Bechet M.
Published: February 2012

Follicles from adult rat thyroid glands were isolated by digestion with collagenase followed by sieving. Electron microscopic studies have shown that basement membrane was present around each one of the isolated follicles. Follicles were cultured in vitro with or without thyrotropin and fibronectin. When cultured in a medium free of thyrotropin, basement membrane remained around them, while it was lacking in presence of this hormone.
An ultrastructural and cytochemical study of the nucleolus‐DNA body complex of Amphiporus lactifloreus oocytes
Authors: Rué G. , Bierne J.
Published: February 2012

The components of the nucleolus‐DNA body complex of oocytes from the hoplonemertean, Amphiporus lactifloreus, were studied by electron microscopy and cytochemistry. In the early stages of meiotic prophase I, the nucleus of female germ cells was characterized by a large heterochromatic body. From this body a polymorphic nucleolar apparatus, the nucleolus‐DNA body complex, subsequently arose. This nucleolar complex was made up of spherical components termed nucleolar spherulae, which contained fibrillar electron‐opaque material with cortical 20 nm dense granules, and characteristic ribbon‐shaped components termed nucleolar lamellae also with dense granules. The latter components originated from the former by a process of self‐peeling of the spherula cortex. A high level of transcriptional activity was located in the nucleolar spherulae which also contributed with the nucleolar lamellae to the formation of many secondary nucleoli and granular bodies. By ultrastructural cytochemistry a number of DNA‐containing structures were visualized in nucleolar areas: first, cytochemically well‐characterized DNA of partly decondensed chromatin was demonstrated in the thick cortex of spherulae and the inner part of lamellae arising from spherula cortex; second, more dispersed chromatin fibers, related with the cortical component, were revealed in the core of nucleolar spherulae, where ribonucleoproteins were also seen. A thin layer of ribonucleoproteins was also detected in the edge of both spherulae and lamellae of the nucleolar complex. These findings, which show that the nucleolar complex of previtellogenic oocytes of hoplonemerteans is basically dual, are discussed in terms of two synchronous functions: a) the start of the transcription in the nucleolus, b) dispersal of the intranuclear rDNA as a prerequisite to subsequent nucleolar transcription activity.
Ultrastructural aspects after cryoultramicrotomy of bone tissue and sutural cartilage in neonatal mice calvaria
Authors: Boivin G. , Morel G. , Meunier P. J. , Dubois P. M.
Published: February 2012

Using transmission electron microscopy after cryoultramicrotomy, mineralized as well as unmineralized bone tissues and sutural cartilage were observed in neonatal mice calvaria. A good definition of osteoblasts (nucleus, membranes, organelles) and extracellular constituents (collagen fibrils, matrix vesicles, mineral substance) was obtained. The sutural zone was composed of an unmineralized cartilaginous tissue with more or less hypertrophic cells surrounded by a finely fibrillar matrix.
Morphometry of fetal Leydig cells in the monkey (Macaca fascicularis), correlation with plasma testosterone
Authors: Fouquet J. P. , Meusy‐Dessolle N. , Dang D. C.
Published: February 2012

The evolution of Leydig cells in Macaca fascicularis fetuses was followed throughout gestation (50‐150 d) by morphometric procedures (volume densities of: cells, SER, mitochondria and lipid droplets). Testosterone from umbilical artery plasma was radioimmunoassayed starting on day 57. After predifferentiation and differentiation phases, Leydig cells entered the maturity phase (57‐66 d), they occupied 19% of testicular volume, SER and lipid droplets represented 19% and 5% respectively of cytoplasmic volume. Then Leydig cells regressed dramatically (involution phase I: 66‐83 d), their volume density decreased to 8%, that of SER to 12% whereas lipids doubled. Leydig cell volume density diminished to 5% during the second half of gestation (involution phase II), but their ultrastructure was not significantly altered. High plasma testosterone level (2.4 ng/ml) was observed during the maturity phase of Leydig cells, decline of testosterone occurred during involution phases I and II (1.13 and 0.58 ng/ml respectively). Its was shown that from day 57 to the end of fetal development the evolution of the plasma testosterone level correlated with the Leydig cell volume density and the SER volume density.
Pathology of the cytoskeleton of the human sperm flagellum: axonemal and peri‐axonemal anomalies
Authors: Escalier D. , David G.
Published: February 2012

A quantitative ultrastructural study was performed on 56 ejaculates showing anomalies of the sperm axonemal complex. The anomalies comprised either the absence of one, or more often several, axonemal structures, or defective elongation of the doublets. Several characteristics relating to the extent and superimposition of the various anomalies could be described and enabled the definition of 6 groups of anomalies. In decreasing order of frequency these were: absence of the doublets and peripheral junctions, absence of the central complex, of the outer dynein arms, of the central junctions, of both dynein arms, and absence of the inner dynein arms and peripheral junctions. Some anomalies caused total immobility, whereas others caused abnormal movement patterns. Abnormalities of the peri‐axonemal structures were found in each group. The various light microscopic characteristics of each of the 6 groups represented 6 seminal profiles which should permit their detection during a routine semen analysis. Several specific associations of axonemal and/or peri‐axonemal anomalies would suggest some morphogenetic links between them. Relationships between the absence of doublets or the absence of the central complex and disturbances of microtubular polymerization are discussed. Finally, the study has provided new data on the composition of the axoneme.
Role of the gonad cytoplasmic core during oogenesis of the nematode Caenorhabditis elegans
Authors: Gibert M. A. , Starck J. , Beguet B.
Published: February 2012

In order to elucidate the function of the cytoplasmic core (or rachis: a structure specific of the nematode gonads), we have carried out a cytological study of this structure in the free‐living nematode Caenorhabditis elegans, in wild‐type and in several mutant strains showing an abnormal gametogenesis. We also performed an ultrastructural radioautographic study of RNA synthesis during oogenesis in order to examine the part played by the rachis in the transport of nutritive substances. Our results evidence for the first time a metabolite transfer from the germ cells to the cytoplasmic core and lead us to assign to the core a trophic role linked to oogenesis. A statistical analysis of silver grain distribution has led us to conclude that there is no accumulation of RNA labelling in any part of the cytoplasmic core. In addition, our studies performed on sterile mutant strains suggest that the cytoplasmic core may have a specific function in oogenesis determination.
Terminal differentiation of hemopoietic cell clones cultured in tridimensional collagen matrix: in situ cell morphology and enzyme histochemistry analysis
Authors: Lanotte M.
Published: February 2012

Collagen, a major component of the extracellular matrix in vivo, has been used as a tridimensional gel matrix for cultured hemopoietic clones. Its resemblance to the natural matrix produced by cells makes it ideal for studies on proliferation and differentiation of hemopoietic lineages. Every lineage, including granulocytes (basophilic, eosinophilic and neutrophilic polymorphs) monocyte‐macrophages, megakaryocytes, erythroid and lymphoid lineages could be grown using a standardized collagen medium, provided that specific stimulators were added in the culture. Clones were scored on either live or fixed cultures. Compared to other gel substrates, collagen matrix proved superior for cell proliferation and maturation. Additional advantages (in situ clonal analysis by histological staining, enzyme cytochemistry), and other possibilities of the method are reported and discussed. The system offers great potential for cellular immunology, hematology and molecular biology with peculiar reference to differentiation of normal hemopoietic cells, viral transformation and leukemogenesis in vitro. These applications are reviewed.
Electron microscopic study of the development of Theiler's murine encephalomyelitis viruses propagated in vitro
Authors: Friedmann A. , Lorch Y.
Published: February 2012

The changes occurring in the nuclei and cytoplasms of BHK21 cells during infection with the two subgroups of Theiler's murine encephalomyelitis viruses were studied by electron microscopy. The nuclear alterations include: formation of clumps and margination of chromatin, enlargement of perinuclear spaces, deformation and displacement of the nucleus. The cytoplasmic alterations include: increase in the number of ribosomes, extensive proliferation of smooth membranes at the centrosphere of infected cells, appearance of electron‐dense bodies, and decrease in number and swelling of mitochondria. At late stages of infection, GDVII and FA viruses which represent the virulent subgroup, form crystalline arrays in the cytoplasm of infected cells. A few mechanisms by which these crystals may be formed are discussed. The later stages of TO viruses infection, which cause a persistent infection in mice, differed markedly from those of GDVII and FA viruses. TO viruses were found to be arranged in a single file between two sheets of membranes. A model for this unique structure is presented.
Quantitative assessment of the glandular activity of endocrine cells of gastrointestinal mucosae by computer‐assisted analysis of ultrastructural images: methodology
Authors: Pradal G. , Lefranc G. , Mollat F. , Vo H. , Riet A.
Published: February 2012

A method is described for studying the morphological features of endocrine cells of gastrointestinal mucosae in man and some animal species by semi‐automatic analysis of ultrastructural images. The method enables possible ultrastructural variations in gastrointestinal endocrine glandular cells of different types to be determined with an acceptable margin of error. Various causes of error are investigated. It is found that the main cause is the choice of microscope magnification, despite the corrections made. The factor of inconsistency in the reproducibility of measurements performed by several operators is also calculated.
RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study
Authors: Motlík J. , Kopecný V. , Trávnik P. , Pivko J.
Published: February 2012

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5‐0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6‐2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5‐6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver‐positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5‐6 mm.
The periseptal annulus in Escherichia coli
Authors: Anba J. , Bernadac A. , Pages J. M. , Lazdunski C.
Published: February 2012

Evidence is presented that two circumferential zones of cell envelope differentiation, the periseptal annuli, exist in E. coli as previously observed in S. typhimurium. The periseptal annulus is located at the division site of cells. A strain overproducing a periplasmic protein, PhoS (phosphate‐binding protein) has been used to provide a landmark for the periseptal compartment. The zone of adhesion does not involve inner‐outer membrane fusion. This zone does not provide a strong physical barrier to protein diffusion in the periplasmic space, at least under conditions of plasmolysis.
Con A‐ and WGA‐binding sites on bovine epididymal spermatozoa: TEM of specimens in toto
Authors: Friess A. E. , Sinowatz F.
Published: February 2012

Binding sites for Con A and WGA were detected on bovine spermatozoa during epididymal maturation. We used colloidal gold as an EM‐marker. The spermatozoa were treated according to a two‐step method for lectin and colloidal gold, then adsorbed to lysine‐coated nickel grids and subsequently examined by TEM in toto. Using this method we rapidly got information about the topographic distribution of lectin‐binding sites. Major differences exist for WGA between caput and cauda spermatozoa. Conceding that cell‐thickness poses some limitation, we consider this method to be practical and especially useful in studies concerning topographic distribution of cell surface components in single cell systems.
Subcellular responses in frog pituitary gonadotrophic cells to constant environmental conditions: a morphometric study
Authors: Gracia‐Navarro F. , Gonzalez‐Reyes J. A. , Garcia‐Navarro S. , Garcia‐Herdugo G.
Published: February 2012

An ultrastructural study of the pars distalis of the pituitary in Rana ridibunda specimens subjected to constant environmental conditions revealed significant structural differences which indicate a short‐time increase in the activity of gonadotrophic cells. Planimetry was used for morphometric and stereological analysis of the rough endoplasmic reticulum, Golgi complex, secretory granules and lysosomes. The presence of a vesicular endoplasmic reticulum throughout the cytoplasm of gonadotrophic cells was observed in the pituitary gland of the intact frog. After 7 days of constant environmental conditions, there was an increase in the volume density of the endoplasmic reticulum vesicles, while on the 11th day there was a slight reversal in the vesicle content that was complete the 15th day. In the hypertrophy of the endoplasmic reticulum, the larger vesicles (bigger than 0,52 micron2) play a more important role than the smaller ones (similar to the endoplasmic reticulum observed in control cells). However, the small vacuoles were also hypertrophied in relation to the control. Degranulation was also observed after 7 days of constant environmental conditions. On the 15th days, there was an increase in the numerical density of secretory granules and lysosomes compared to the control while their volume density was similar to the control.
Inhibition of the endocytic pathway for asialoglycoprotein catabolism
Authors: Harford J. , Klausner R. D. , Ashwell G.
Published: February 2012

Rat hepatocytes in primary culture bind, internalize and eventually degrade asialoglycoproteins. This process is mediated by a specific receptor in the hepatocyte plasma membrane. The endocytic pathway by which ligand molecules are translocated to lysosomes has been examined by the development of biological assays capable of distinguishing ligand populations at various points in the process. Inhibitors have been identified that perturb particular transitions that define the endocytic pathway. In the present paper, inhibition by the bacterial tripeptide leupeptin is compared to the effect of colchine plus cytochalasin B. The latter combination impedes intracellular segregation of ligand and receptor while leupeptin inhibits intralysosomal proteolysis. However, evidence is presented to indicate that the inhibitory effects colchine plus cytochaasin B consists of at least two components. One component is independent of the presence of ligand whereas the other is observed only when ligand is present together with the drugs.
An endogenous carbohydrate‐binding agglutinin of BHK cells. Purification, specificity and interaction with normal and ricin‐resistant cell lines
Authors: Stojanovic D. , Hughes R. C.
Published: February 2012

Several lectin‐like activities were detected on the surface of unfixed, viable BHK cells by reaction with FITC‐labelled glycosylated albumin derivatives. A prominent surface staining was obtained with the beta‐lactosyl, beta‐N‐acetylgalactosaminyl, alpha‐mannosyl and beta‐N‐acetylglucosaminyl derivatives. Endogenous lectin‐like activities were also detected in BHK cell homogenates by haemagglutination of glutaraldehyde‐fixed rabbit erythrocytes. Haemagglutinating activity was purified by chromatography of sodium deoxycholate‐extracts of BHK cell microsomal fractions on Sepharose 4B and asialofetuin‐Sepharose 4B. A purified agglutinin was eluted from the latter column with 0.2 M thiodigalactoside. The haemagglutination mediated by the purified factor was inhibited by thiodigalactoside, N‐acetylgalactosamine, galactosyl‐beta 1‐4‐N‐acetylglucosamine and several glycoproteins. The purified agglutinin agglutinated trypsinised, fixed normal BHK cells more readily than several ricin‐resistant cell lines. By contrast, a mannose‐binding lectin from rabbit serum reacted equally well with normal and mutant cells. These results are in general agreement with models of cell‐cell adhesion involving the interaction of surface located lectins with carbohydrate sequences of normal BHK cell surface glycoproteins.
The recognition of red blood cells by macrophages: role of phosphatidylserine and possible implications of membrane phospholipid asymmetry
Authors: Schroit A. J. , Tanaka Y. , Madsen J. , Fidler I. J.
Published: February 2012

The recognition of phosphatidylserine (PS) by macrophages was investigated using inside‐out (IO) red blood cell (RBC) ghosts and RBC displaying PS in their surface membranes. This was accomplished by employing unmodified pathologic sickle RBC which contain endogenous PS in their outer membrane leaflet, and RBC modified by the transfer of an exogenous fluorescent PS analog. Proper insertion of exogenous PS was confirmed by monitoring the degree to which cell‐associated lipid fluorescence was dequenched following transfer of 1‐acyl‐2‐[(N‐4‐nitro‐benzo‐2‐oxa‐1,3 diazole) aminocaproyl] phosphatidylserine (NBD‐PS) from a population of self‐quenched donor vesicles. Inside‐out RBC ghosts were endocytosed approximately 3 times faster than were right side‐out control populations. Similarly, using NBD‐PS vesicles at concentrations at which dilution of all the cell‐associated analog in the recipient RBC could be unequivocally confirmed, we observed that the uptake of NBD‐PS treated RBC by macrophages was significantly increased over that of control RBC populations. Fluorescence and electron microscopic observations revealed the formation of typical RBC‐macrophage rosettes that were morphologically distinct from opsonized RBC‐macrophage rosettes. Enhanced RBC binding to macrophages was also obtained with deoxygenated reversibly sickled cells (RSC); the enhancement correlated with increased exposure of outer leaflet PS in these cells. These findings suggest that PS is recognized by macrophages and that its exposure in the outer leaflet of RBC may have significant pathophysiologic implications.
The role of carbohydrate in heterotypic cell‐cell recognition: lymphocyte‐high endothelial cell interaction as a model system
Authors: Butcher E. C.
Published: February 2012

The studies reviewed here demonstrate that the interaction of lymphocytes with HEV is one of the most approachable models available for the study of heterotypic cell‐cell recognition mechanisms. Lymphocyte‐HEV interaction is mediated by specific lymphocyte surface receptors recognizing, by as yet unknown mechanisms, determinants expressed by specialized high endothelial cells in lymphoid tissues and sites of chronic inflammation. Both the lymphocyte and endothelial cell surface elements of the interaction are precisely regulated, controlling the traffic of lymphocyte subsets through particular lymphoid organs and into sites of inflammation. Considerable progress, reviewed here, has been made in defining and characterizing the lymphocyte surface molecules mediating this cellular interaction. By contrast, the nature of the endothelial cell determinants recognized by migrating lymphocytes remains a mystery. Future experiments must be designed to identify these endothelial cell determinants, and to examine critically a proposed role of carbohydrate in lymphocyte‐HEV interaction.
Effect of prolactin on the secretion of milk casein: metabolism of arachidonic acid
Authors: Ollivier‐Bousquet M.
Published: February 2012

Mammary gland fragments were incubated in the presence of prolactin and arachidonic acid which stimulate casein secretion. The effects of these stimuli in the presence of agents that influence arachidonic acid metabolism were investigated. Chloroquine, a blocker of phospholipase A2 activity, decreased prolactin but not arachidonic acid stimulation of casein secretion. Phospholipase A2 markedly stimulated casein secretion. Nordihydroguaiaretic acid (NDGA), an antioxidant that inhibits lipoxygenase, blocked the stimulating effect of prolactin and arachidonic acid. Ultrastructural studies indicated that phospholipase A2‐induced stimulation of secretion was comparable to that of prolactin but that arachidonic acid‐induced stimulation did not involve the same Golgi membrane modifications. These studies suggest that prolactin and phospholipase A2 stimulate secretion by a common way, and that arachidonic acid interferes with secretion by metabolic products of the lipoxygenase pathway.
Opposite effects of glucocorticoid on estrogen‐induced growth and differentiation of quail oviduct: demonstration by sequential treatments
Authors: Boisvieux‐Ulrich E. , Laugier C. , Sandoz D.
Published: February 2012

Control of the development and functions of avian oviduct is monitored by four classes of steroid hormones, including glucocorticoids. The effects of dexamethasone (DEX), a synthetic glucocorticoid, were studied via sequential treatments with estradiol benzoate, paying special attention to changes in estrogenic oviduct responses involving DNA synthesis and cell proliferation, ovalbumin accumulation and cell differentiation. DEX exerted an antagonistic effect upon estrogen stimulation when administered separately before or after estradiol benzoate (EB). Given before EB, DEX was more strongly antagonistic for DNA synthesis than when given simultaneously with EB. Administered after EB, DEX reversed EB‐induced cell proliferation: the DNA content declined and the oviduct regressed. In the same way, protein and ovalbumin synthesis was inhibited and delayed by first intervention of DEX, and accelerated catabolism of ovalbumin and proteins was observed when DEX followed EB. DEX, which was ineffective alone, but synergistic on ovalbumin synthesis when given concomitantly with EB, prevented or dissipated the estrogenic effects, cell proliferation and secretory process when administered in sequential treatments.
Correlation between changes in cell adhesion and the ratio of N‐ to O‐linked glycopeptides during chick embryo development
Authors: Berjonneau C. , Aubery M. , Vernay M. , Bourrillon R.
Published: February 2012

After treatment with trypsin, chick embryo fibroblasts exhibited an age‐related difference in their capacity to readhere to the substratum, since 8‐day‐cells readhered more rapidly than 16‐day‐cells. Treatment with tunicamycin altered embryo cell readhesion to the substratum in varying degrees, depending on the duration of drug treatment and of readhesion assay. The effect of tunicamycin was not toxic and was totally reversible with time after its removal. These results indicated that embryo cell readhesion involved trypsin‐sensitive cell surface glycoproteins. During embryo development, the glycosylation of cell surface glycoproteins altered markedly. The ratio of N‐linked to O‐linked glycan chains dropped from 80/20 in 8‐day‐cells to 55/45 in 16‐day‐cells, indicating that the relative labelling of O‐linked glycan chains increased during embryo development. This result was confirmed by alkaline treatment of radiolabelled glycan chains, and by the fact that tunicamycin treatment reduced 14C‐glucosamine incorporation by greater than or equal to 80% in 8‐day‐cells but only 60% in 16‐day‐cells. Marked changes were observed during embryo development in the structure of the N‐linked glycan chains; concanavalin A‐Sepharose chromatography showed that these changes concerned the glycopeptides containing complex type carbohydrate chains. The ratio of tri‐ plus tetra‐antennary chains to bi‐antennary chains increased about 2.5‐fold between the 8th and 16th day of development. A correlation was noted between embryo cell readhesion to the substratum and N‐glycosylation of cell surface glycoproteins. The N‐linked glycoconjugates played a crucial part in cell readhesion. The possible role of O‐linked structures in such readhesion is discussed.
Specific binding and biological effects of tumor promoting phorbol esters on sponges
Authors: Mazzorana M. , Garrone R. , Martel N. , Yamasaki H.
Published: February 2012

Sponges grown in the presence of 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) show deep alterations of their structure and development. Their aquiferous system (flagellated cells and canals) is largely altered and the tissues show an unusually high cell density. This focalized effect of TPA on the aquiferous system seems specific and is reversible at low concentrations (100 ng/ml). A toxic, non‐specific effect is also noted, particularly at high concentrations (5000 ng/ml). Using 3H‐phorbol‐12, 13‐dibutyrate (3H‐PDBu), we demonstrate a class of specific binding sites for phorbol esters in the homogenates of sponges. These binding sites have high affinity (Kd = 26.0 nM) for PDBu and at saturation about 20 pmoles of 3H‐PDBu is bound per mg protein of sponge homogenates. The binding of 3H‐PDBu was inhibited by other phorbol esters and their congeners, and there was a good correlation between their potency in binding inhibition and their tumor promoting activity. It is concluded that sponges have a class of specific saturable and high affinity receptors for phorbol esters and that there is a very high conservation of these receptors during evolution. Such specific binding may be responsible for subsequent biological effect of TPA on sponges.
Study of a lysis medium stabilizing microfilaments and microtubules in vitro and in vivo
Authors: Foucault G. , Raymond M. N. , Coffe G. , Pudles J.
Published: February 2012

Determination of experimental conditions which allow the evaluation of the variations in the ratio of non polymerized and polymerized forms of actin and tubulin during the reorganization of the cytoskeletal cell system is of most valuable importance. In order to prepare cell homogenates which would reflect the in vivo situation, we tested in vitro a lysis medium which stabilized both microfilaments and microtubules, which were determined by DNase inhibition assays and colchicine binding assays respectively. This lysis medium containing 10 mM potassium phosphate, 1mM magnesium chloride, 5 mM EGTA, 1 M hexylene glycol, 1% Triton X‐100, pH 6.4, used at 4 degrees C a) diffused rapidly into the cells; b) did not denature actin and tubulin; c) did not displace the equilibrium between non polymerized and polymerized forms of actin and tubulin, allowing biochemical assays on cell homogenates; d) blocked the evolution of the cytoskeletal system and permitted structural studies; e) and allowed the decoration of microfilaments by heavy meromyosin.