摘要:

Summary—Olomoucine (2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine) has been recently described as a competitive inhibitor (ATP‐binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5p35 kinase and the ERK1AP‐kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1S transition of unicellular algae (dinoflagellate and diatom). It blocksFucuszygote cleavage and development ofLaminariagametophytes. StimulatedPetuniamesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development ofCalanuscopepod larvae. It reversibly inhibits the early cleavages of Caenorhabditiselegansembryos and those of ascidian embryos. Olomoucine inhibits the serotonin‐induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists.Xenopusoocyte maturation, thein vivoandin vitrophosphorylation of elongation factor EF‐1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF‐7, KB‐3‐1 and their adriamycin‐resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non‐small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin‐2‐stimulated T lymphocytes (CTLL‐2 cells) and triggers a G1 arrest similar to interleukin‐2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole‐ and hydroxyurea‐treated CTLL‐2 cells, respectively) are inhibited by olomoucine. Both yeast andDrosophilaembryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1S and the G2M boundaries, consistent with the hypothesis of a prevalent effe

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81298-6

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—While it is known that the small GTP‐binding protein rab6 is localizedin vitroto the Golgi apparatus of several mammalian cells, its behaviourin vivohas not yet been investigated. The aim of this work was to compare by immunocytochemistry and immunoblotting the distribution of rab6 in hepatocytes from normal rats and from rats with an acute inflammatory reaction, a circumstance where the synthesis and secretion of plasma proteins by the hepatocytes is increased and which is accompanied by several changes in the Golgi apparatus. Our results show that in normal rats, rab6 was present in all hepatocytes irrespective of the location of the cell in the hepatic lobule. At the ultrastructural level, rab6 was mainly visible on the three Golgi saccules, but in some cells it appeared to be absent in saccules corresponding tothe cisor thetranssaccule. The inflammatory reaction was accompanied by an increase of the immunocytochemical labelling at the light and electron microscopy levels. However, by immunoblotting, no differences in the total amount of rab6, nor in its subcellular distribution were found in liver cells after acute inflammatory reaction. These results demonstrate that rab6 is restrictedin vivoto the Golgi apparatus and that no significant redistribution occurs during an acute inflammatory react

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81299-8

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—The effects of nicotinamide on hepatocyte viability and secretion of albumin and α1‐acid glycoprotein were studied in the absence or presence of dexamethasone and/or recombinant human interleukin‐6 either after cell attachment (2 h) or after 24, 48, and 72 h of culture. The evolution of hepatocyte survival during the culture was appreciated by measurement of total DNA content. The secretion of albumin and α1‐acid glycoprotein was measured after a 4‐h period following cell attachment or after 24, 48 and 72 h of culture. The important decrease of DNA content, mRNA levels and secretion of albumin and α1‐acid glycoprotein in control cultures after 2–3 days was not prevented by the addition of nicotinamide. In contrast, dexamethasone alone or with recombinant human interleukin‐6 improved DNA content and albumin secretion with no additional effect of nicotinamide. The secretion of α1‐acid glycoprotein was largely induced by dexamethasone alone or dexamethasone and recombinant human interleukin‐6. The increase of α1‐acid glycoprotein secretion was not modified by the addition of nicotinamide and averaged respectively 27‐ and 60‐fold for dexamethasone alone and dexamethasone and recombinant human interleukin‐6 after 48 h. These observations suggested that nicotinamide, at least in the conditions tested here, is unable to prevent alterations of hepatocyte viability and gene ex

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81300-1

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Muscular dysgenesis is a mutation which is characterized by paralysis of skeletal muscle cells. Excitation‐contraction coupling is deficient and muscle cells display atypical ultrastructure.In vitro, mutant myotubes recover a normal phenotype when cocultured with spinal cord cells from normal animals or with normal fibroblasts. We have shown that other types of cells,egcertain glial cells present in the spinal cord or in other tissues, have this capacity. In contrast, intervention of neurons in the recovery does not appear likely. Very different types of non‐myoblastic cells, then, are capable of restoring contractile activity of dysgenic myotubesin vitro, suggesting that a non‐specific mechanism is involved in the phenotypic reversion of affected muscle cells. The restoration process seems to imply a close relationship between myotubes and normal glia

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81301-3

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—This study deals with the acid phosphatase (AcPase) of the Kurloff body (KB), a large (10 μm diameter) periodic acid‐Schiff‐positive lysosomal inclusion body present in Kurloff cells (KC). KC AcPase were extracted, with similar yields, either with non‐ionic detergent solution or after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase. After DEAE‐cellulose chromatography of such crude Dounce‐extracts, 97% of KC AcPase activity was recovered in the unbound glycoprotein fraction (peak I). The main protein content consisted of, as testified by SDS‐PAGE analysis, major KC glycoproteins of 30–35 kDa. Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to theseN‐glycosylproteins among which the presence of α (2,6) sialoglycoproteins was previously established. Following electrophoresis on a 4–15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations (200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectively detected by zymography in peak I. AfterClostridium‐derived sialidase digestion of peak I, the highly active bands observed at pH 3.5 −5.2 disappeared. These zymographic patterns were similar to those obtained with crude extracts. After Concanavalin A (ConA)‐Sepharose chromatography of peak I, the single ConA‐bound glucosamine‐labelled peak, eluted at 200 methyl‐α‐d‐mannopyranose, contained the AcPase activity while the ConA‐unbound peak was devoid of any acid phosphatase activity. After SDS‐PAGE analysis, the ConA‐bound fraction appeared to correspond only to a single broad protein band in the 30–35 kDa zone. The Sambucusnigraagglutinin‐reactivity of the latter band confirmed the α (2,6) sialylation pattern of KC AcPase. Moreover, their strong binding to ConA‐Sepharose suggests that they could only correspond to the hybrid type of theN‐glycosylproteins, since they could not correspond to the oligomannosidic type considering the abse

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81302-5

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—One of the initial events required for the expression of cartilage‐specific macromolecules in monolayer cultures is the reversion to the initial round shape of chondrocytes. Thus, considerable research efforts have focused on developing reliable procedures to maintain a round morphology of cultured chondrocytes. Our study focuses on evaluating the response of dedifferentiated fetal rat chondrocytes to cytochalasin D, an actin‐disrupting agent, with special emphasis on the morphological events. Immediately after exposure to the drug, cells round up but flatten again after removing the agent. However, immunocytochemical procedures revealed a disorganization of microfilaments and intermediate filaments. Phase‐contrast and scanning electron microscopic observations revealed that on day 6 of culture, cells located at the top of the cell layer adopted a spherical morphology. Prominent differences were noted in control cultures where cells had to aggregate prior to overt chondrogenesis. Transmission electron microscopy confirmed the round morphology of the cells situated at the top layer but also revealed the presence of cell contacts between the cells. In addition, cells located at the central part of the cell layer displayed a typical morphology of mature chondrocytes, separated by an extensive extracellular matrix. These morphological changes occurred parallel to the expression of type II collagen and chondroitin sulfate, both hallmarks of the chondrocyte phenotype strong in experimental cultures, relatively weak in control cultures, and only restricted on areas of polygonal cellular aggregates. Furthermore, [35S]‐sulfate incorporation into sulfated glycosaminoglycans increased rapidly with the period of culture to a maximum after 7 days and was then two‐fold in treated cultures. Taken together, these findings indicated that cytochalasin D stimulates chondrogenesis in response to modification of cytoskeleton architecture and the subsequent rounding up o

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81303-7

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—The modification induced by interferon (IFN) in brown adipose tissue (BAT) was studied by high spatial resolution magnetic resonance imaging (MRI), histology, and transmission electron microscopy (TEM). In IFN‐treated mice, at MRI, the interscapular BAT was slightly enlarged and showed non‐homogeneous areas of lipid accumulation. The thickness of the subcutaneous white adipose tissue was reduced with respect to control mice. In the liver, MRI showed a lipid accumulation. In IFN‐treated mice, by light microscopy, brown adipocytes showed a larger lipid deposit with respect to control mice. At TEM, in BAT, the mitochondria were reduced in number, smaller and the number of cristae was also significantly reduced with respect to the controls (9.1 ± 1.5 vs20.1 ± 1.9, P<0.01). The inclusions in the mitochondrial matrix were significantly less numerous in IFN‐treated than in control animals (1.9 ± 0.7 vs0.9 ± 0.7 for mitochondrial section, P<0.01). Abnormalities of endoplasmic reticulum described in hepatocytes were not found in brown adipocytes of IFN‐treated mice. The present work demonstrates that, in the BAT of sucking mice, IFN‐treatment induces morphologic alterations and that brown adipocytes have MRI and TEM features resembling those found in the lipid laden BA

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81304-9

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Porcine granulosa cells cultured in serum‐free medium undergo metabolic and morphologic changes after follicle stimulating hormone (FSH) stimulation. Under these conditions, granulosa cells differentiate and tend to round up and their links with the plastic support are reduced. Coating of culture substratum with PepTite‐2000, an integrin‐binding synthetic peptide containing RGD (Arg‐Gly‐Asp) sequences enhanced the plating of granulosa cells. Whether the peptide be present or not, cells cultivated in basal synthetic medium (without FSH) were flattened and attached to the substratum by stress fibers at focal contacts where integrin β1, extracellular fibronectin, and urokinase plasminogen activator colocalized. After FSH stimulation, part of the cells rounded up and F‐actin took a more uniform, cortical localization. Correlatively, extracellular fibronectin aggregated in a clump, while integrin β1 and urokinase plasminogen activator spread over rounded cells. These morphological changes elicited by FSH were little affected by the presence of PepTite‐2000, yet a larger number of cells remained flattened. However, concerning steroidogenesis, increasing concentrations of peptide seemed to favor progesterone rather than estrogen production, and to restrain luteinizing hormone (LH) receptor expression, suggesting a premature committment of cells towards luteinization rather than completion of follicular preovulator

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81305-0

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—The specificity of four monoclonal antibodies, raised against mammalian ciliary axonemes, was determined by both immunofluorescence and immunoblot experiments. Three antibodies reacted with epitopes which are differentially located along axonemal length. Among these, antibody 3.12 recognized an epitope common to different dynein heavy chains, reacted only with tracheal cilia and specifically stained the proximal portion of the ciliary axonem

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81306-2

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Employingin situnick translation on isolated macronuclei from the ciliate Euploteseurystomus, the replication band exhibited heightened DNase I sensitivity. This enhanced reactivity was not significantly affected by decreased replication induced with heat shock or added inhibitors, implying that the underlying chromatin modulations are reasonably stable. The nuclease sensitivity of the replication band is probably related to the ‘maturation’ of nascent chromatin, since transcriptional activity is known to be absent within the band. An additional observation of this study was thein vivoincorporation of halogen‐deoxynucleosides into replication bands, facilitating future studies of matrix‐associated na

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)81307-4

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY