Cytoplasmic Matrix Research Articles

Mechanisms of Heat-induced Antigen Retrieval: Does pH or Ionic Strength of the Solution Play a Role for Refolding Antigens?

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.

Application of Heat-induced Antigen Retrieval to Aldehyde-fixed Fresh Frozen Sections

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl(2) for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120 C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl(2), 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERalpha immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl(2) at 40 C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

Cellular and stromal characteristics in the scirrhous hepatocellular carcinoma: Comparison with hepatocellular carcinomas and intrahepatic cholangiocarcinomas

Scirrhous hepatocellular carcinoma (SHCC) is a rare variation of HCC, for which characteristics of tumor cells and the fibrotic stroma have not been clarified in detail. The present study was therefore carried out to elucidate cytological features of tumor and stromal cells and components of the stromal extracellular matrix in 15 SHCC patients undergoing hepatectomy without preoperative transarterial embolization. Diagnosis was on the basis of a scirrhous histological pattern exceeding 50% of the tumor area. Expression of cytoplasmic and extracellular matrix proteins was compared among SHCC, HCC and intrahepatic cholangiocarcinoma (ICC) cases with immunohistochemical staining. The lesions could be histologically divided into radiating and sinusoidal types. Common stromal components of SHCC and ICC were collagen types I and III. There was no expression of laminin-5 in the stroma of SHCC, but it was present in almost all ICC cases. Tenascin-C expression was significantly lower in the SHCC cases and its distribution differed between SHCC and ICC. Matrix metalloproteinase-7 (MMP-7) expression was significantly higher in SHCC compared with HCC. Almost all stromal cells were alpha-smooth muscle actin-positive both in SHCC and ICC, whereas glial fibrillary acid protein (GFAP)-positive stromal cells were significantly more increased in ICC than in SHCC. SHCC clearly differed from HCC with respect to collagen types I, III and MMP-7 expression, and from ICC with regard to stromal components including laminin-5, tenascin-C and GFAP(+) stromal cells.

Quel rôle spécifique pour ANT2 dans une cellule cancéreuse ?

In the mitochondrial internal membrane, the adenine nucleotide translocator (ANT) carries out the ATP/ADP exchange between cytoplasm and mitochondrial matrix. Three isoforms with different kinetic properties are encoded from three different genes in Human: the muscle specific ANT1 and the ubiquitary ANT3 isoforms export ATP produced by mitochondrial oxidative phosphorylation (OXPHOS). The ANT2 isoform is specifically expressed in proliferative cells with a predominant glycolytic metabolism and is associated with cellular undifferentiation which is a major characteristic in carcinogenesis. Its role would be to import into mitochondria ATP produced by the glycolysis, energy essential to several intramitochondrial functions, particularly to maintenance of the membrane potential (Delta Psi m), conditioning cellular survival and proliferation. The mechanism of regeneration of this Delta Psi m gradient would involve at least three major proteins: the hexokinase II isoform, the ANT2 isoform and the F1 part of the mitochondrial ATP synthase complex. Taking into account this major role of ANT2 in cell proliferation and the very low expression of this isoform in differentiated tissues, this protein or its transcript could be chosen as a target for an anticancer strategy. Furthermore, previous studies showed that molecules of the cisplatin family, used as chemotherapeutic agents, led to the destruction of the mitochondrial membrane potential and thus to cell death. Does the anticancer effect of these molecules result, at least partially, from this mitochondrial aggression? If it is the case, the ANT2 isoform, mainly involved in the generation of this potential by its ATP4-/ADP3- exchange, could be considered as a more specific targeting by an RNA interference approach.

Ontogeny of expression of transforming growth factor‐β and its receptors and their possible relationship with scarless healing in human fetal skin

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-beta (TGF-beta) isoforms and their receptors in the wound healing process, their roles in scarless wound repair observed in early gestation and their functions in human fetal skin development, and structural and functional maintenance are still not well understood. In this study, we explore the expression and distribution characteristics of three TGF-beta isoforms and their receptors, TGF-betaRI (TBRI) and TGF-betaRII (TBRII), in fetal and postnatal skins to understand the relevance of these five proteins to skin development and elucidate the mechanism(s) underlying the phenotypic transition from scarless to scar-forming healing observed during fetal gestation. Fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages from 13 to 32 weeks and postnatal skin specimens were collected from patients undergoing plastic surgery. Gene expression and positive immunohistochemical signals of TGF-beta(1), TGF-beta(2), TGF-beta(3), TBRI, and TBRII could all be detected in fetal and postnatal skins. In early gestation, gene expression of TGF-beta(1), TBRI, and TBRII was weaker and protein contents were less compared with postnatal skins (p < 0.05). In contrast, more TGF-beta(2) mRNA transcript was found in early gestation than in late gestation and in postnatal skins, whereas protein content of this growth factor increased during gestation. Lastly, mRNA transcript and protein contents of TGF-beta(3) were apparently higher in early gestation compared to postnatal skin (p < 0.05). In postnatal skin, granules containing the three TGF-beta isoforms were mainly distributed in the cytoplasm and extracellular matrix of epidermal cells, interfollicular keratinocytes, and some fibroblasts. TBRI and TBRII were chiefly located in the cellular membrane of epidermal keratinocytes and some fibroblasts. The endogenous three TGF-beta isoforms and their receptors may be involved in the development of embryonic skin and in the maintenance of cutaneous structure and function, and also in postnatal wound healing. The differential levels of TGF-beta isoforms may provide either a predominantly antiscarring or profibrotic signal upon wounding depending on the gestational period. Lower expression of their receptors in early gestational skins may be a reason for the reduced ability to perceive ligands, ultimately leading to scar-free healing.

Impairment of Nuclear Pores in Bovine Herpesvirus 1-Infected MDBK Cells

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.

Destruction of Pigments and Ultrastructural Changes in Cyanobacteria during Photodamage

Sequential stages of pigment degradation and ultrastructural changes were examined in cyanobacteria Anabaena variabilis ATCC 29413, Synechococcus sp. PCC 6301 (Anacystis nidulans) and S. elongatus B-267 during irradiation of cell suspensions with high-intensity light. Early manifestations of photooxidative destruction were evident as profound changes in ultrastructure of thylakoids; in A. variabilis these changes appeared even before bleaching of pigments. Concomitant to these alterations, the cytoplasmic matrix turned homogenous and the nucleoid was subject to degradation, while ultrastructural changes of cytoplasmic membrane and cell walls became evident in some species. In A. variabilis these changes were related to a subsequent autolysis of cells. Synechococcus strains demonstrated comparatively high resistance to irradiation. The experimental data were compared with previously described behavior of the same species of cyanobacteria cultured under photooxidative conditions. This comparison revealed principal similarity and species-specific features in the destructive changes of thylakoids and other cell components of cyanobacterial cells.

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory. X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/ B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/ B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/ B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/ B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/ B ratios at 300 kV. From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/ B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.

Adherence of Staphylococcus intermedius to canine corneocytes involves a protein–protein interaction that is sensitive to trypsin but resistant to cold

Adherence is a prerequisite for canine cutaneous bacterial infection that is potentially affected by a large number of variables. The aim of this study was to explore the nature of this interaction by investigating the effects of long‐term storage, cold temperatures and trypsin on staphylococcal adherence to canine corneocytes. Sheets of corneocytes were collected using double‐sided tape from the ventral abdomen of healthy dogs at 5, 3 and 2 months before use and stored at –80°C, 4°C and room temperature (approximately 20°C) in dry and dark conditions; corneocytes were also collected on the day of each experiment. Staphylococcus intermedius froma case of bacterial pyoderma was prepared in phosphate‐buffered saline (PBS) and applied in triplicate to the canine corneocyte‐covered tapes using PBS as the negative control. After incubation, rinsing and staining with crystal violet, quantification of the adherent bacteria was carried out blindly by computerised image analysis of 15 fields. All the experiments were processed in two duplicate, identical experiments. There were no significant differences in the adherence of S. intermedius to fresh corneocytes and to those that had been stored at –80°C (P = 0.3703) or 4°C (P = 0.1129) at any time point and by subanalysis (P &gt; 0.05 in each case). By contrast, the adherence to corneocytes stored at room temperature showed significant variation (P = 0.0079), with the values for fresh samples being higher than those stored for 5 months; however, subanalysis did not reveal significant differences between individual time points (P &gt; 0.05). These results indicate that the mechanisms of adherence are not affected by even prolonged storage at cold temperatures. Such storage greatly facilitates the performance and comparison of adherence studies. To determine if adherence involved interaction of proteins on the surface of the organism with those on corneocytes, trypsin, a potent broad‐spectrum proteinase, was added to the incubation buffers. Preliminary experiments showed that trypsin concentrations of 1, 10–2, 10–4 and 10–6% did not affect the viability of S. intermedius in incubations lasting up to 4 h. Subsequent adherence assays were therefore conducted using a 4‐h incubation period and the above concentrations. Adherence of S. intermedius to canine corneocytes was completely abolished following incubation of the organism with trypsin at concentrations ranging from 1 to 10–2%, but adherence still occurred at the lower concentrations, demonstrating a dose–response phenomenon. These results suggest that a protein receptor on the surface of the organism was digested, thus eliminating the capacity for adherence. Paradoxically, pretreatment of the corneocytes with trypsin resulted in a significant enhancement in adherence of the organism (P &lt; 0.0035). This may result from exposure of surface receptors after digestion of surface proteins. Alternatively, trypsin may damage the corneocyte envelope and expose cytoplasmic keratin fibres and matrix which express greater affinity for the bacterium. These studies provide evidence that adherence of S. intermedius to canine corneocytes involves a specific protein–protein interaction. Elucidation of the precise proteins involved could provide molecules that might be amenable to therapeutic targeting. Funding: Self‐funded.

Cataract formation in a strain of rats selected for high oxidative stress

The primary purpose of this study was to define the clinical and morphological features of cataractogenesis in the OXYS strain of rats that generate excess reactive oxygen species. Rats were sequentially examined from birth to the development of mature cataracts with slit lamp biomicroscopy. Morphology of selected stages of cataract development was studied using light and transmission electron microscopy (TEM), immunohistochemical localization of the lipid peroxidation product 4-hydroxynonenal (HNE) and fluorescent antibody labeling for DNA oxidation products. Lenses from age-matched normal rats were used as controls. OXYS rats developed cataracts as young as two weeks of age with progression to maturity by 1 year. Clinically, cataracts appeared initially either as nuclear or sub-capsular cortical changes and progressed to pronounced nuclear cataracts within months. TEM confirmed the light microscopic impression of region-specific alterations in both fiber cell cytoplasmic protein matrix and membrane structure. The outer adult nuclear region showed extensive cellular damage similar to osmotic cataracts, which is consistent with the postulated high uptake of glucose in the OXYS strain. The adult and outer fetal nuclear cells displayed several types of focal damage. The inner fetal and embryonic nuclear cells demonstrated textured cytoplasm, suggesting protein degradation or redistribution. Staining for HNE was increased in epithelium, cortex and nucleus compared to control lenses. Fluorescent antibody probes demonstrated increased levels of DNA oxidation products in OXYS rat lenses compared to age-matched controls. Fourier analysis of nuclear cytoplasm revealed significant components with corresponding sizes greater than 100 nm and, using a new theoretical approach, the texturing of the cytoplasm was shown to be sufficient to cause opacification of the nucleus. The OXYS rat appears to be an ideal model for oxidative stress cataractogenesis. The potential oxidative damage observed is extensive and characteristic of the developmental region. The source of oxidative damage may in part be a response to elevated levels of glucose. Because oxidative stress is thought to be a major factor in cataract formation in both diabetic and non-diabetic aging humans, this animal model may be a useful tool in assessing efficacy of antioxidant treatments that may slow or prevent cataract formation.

ISOLASI DAN ANALISIS GEN YANG RESPONSIF TERHADAP CEKAMAN KEKERINGAN PADA TEBU

This study was aimed to isolate and identify a gene that was responsive to drought stress in sugar cane variety M442-51, a variety that is widely used in pastures or dry land. Under drought stress, M442-51 showed an enhance expression of several proteins. One of the protein, namely dip22, (drought inducible protein, with a molecular weight of 22KDa) was isolated and analyzed. Amino acid sequence of this protein showed some homology with several stressed related proteins (OsAsr 1 from Oryza sativa leaves, ZM Bss1 of Zea mays leaves, LeAsr 1, LeAsr 2 and LeAsr 3 of Lycopersicum lycopersicon leaves). The 343 bp of dip22-cDNA fragment was obtained using RT PCR from mRNA of sugar cane leaves that has been exposed to drought stress. The results indicated that the gene dip22 plays an important role as a regulatory protein that are usually nested within cytoplasmic matrix or in the nucleus.

Secreted frizzled-related protein 2 (SFRP2) is highly expressed in canine mammary gland tumors but not in normal mammary glands.

Canine mammary gland tumor (MGT) is the commonest tumor in female dogs and a good animal model of human breast cancer. A group of newly identified genes encoding secreted frizzled-related proteins (SFRP) have been implicated in apoptosis regulation and tumorigenesis. Canine mammary tissues from 50 spontaneous MGTs and 10 normal mammary glands (MGs) were obtained from surgically excised specimens and analyzed for expression of SFRP2, beta-catenin, and cyclin D1. By RT-PCR and in situ hybridization, SFRP2 gene was found abundantly expressed in neoplastic mammary tissues but not in normal mammary tissues, suggesting that SFRP2 may contribute as a tumor marker in canine MGTs. By immunohistochemical staining, the immunoreactivity of the SFRP2 protein was detected in more diverse areas than SFRP2 mRNA expression, including nuclei or/and cytoplasm and extracellular matrix of the tumor. In tumor masses, beta-catenin lost its tight association with the membrane and diffused into the nucleus. The expression of beta-catenin (79.4% positive) and cyclin D1 (71.4% positive) was also increased in MGTs. In the course of tumor progression, SFRP2 mRNA ( p < 0.05) and beta-catenin protein ( p < 0.01) steadily increased but not in cyclin D1. The level of SFRP2 was linearly correlated with its downstream target beta-catenin ( p < 0.05), but not correlated with cyclin D1 ( p < 0.5). As revealed in this study, the exclusive overexpression of SFRP2 in canine MGTs suggests that SFRP2 is a potential candidate gene for further investigation of mammary tumorigenesis and complex etiology of the canine model of mammary neoplasms.

Diamagnetic Susceptibility and Root Growth Responses to Magnetic Fields in Lens culinaris, Glycine soja, and Triticum aestivum

We studied the response of root growth to different magnetic fields and forces. We submitted the seeds of three plant species, Lens culinaris L., Glycine soja Siebold & Zucc., and Triticum aestivum L., which differ in concentrations of paramagnetic (e.g., Fe or Co) and diamagnetic materials (e.g., starchy amyloplasts), to different static magnetic fields and forces. A magnetic field of 176 G reduced root growth of L. culinaris, G. soja, and T. aestivum, 37, 31, and 15%, respectively. A weaker magnetic field of 21 G reduced root growth of L. culinaris and G. soja only 13 and 21%, respectively, whereas it had no significant effect on the cereal T. aestivum. The germinating seeds of L. culinaris and G. soja were less diamagnetic than T. aestivum, and the latter had a smaller paramagnetic component. Since at room temperature, the paramagnetic component was much smaller than the diamagnetic one, the magnetic inhibition of root growth may be linked to the diamagnetic susceptibility, the inhibition being greater for the less diamagnetic materials and for the stronger magnetic forces. These results provide new examples of possible species-specific effects of moderate magnetic fields on plant growth, especially when growth is rapid, such as root growth after germination. We propose a simple hypothesis to relate root growth inhibition with magnetic fields and with the different responses found among species, seasons, and physiological and environmental conditions reported here and in the literature. It is based on a reduced magnetic force acting on the cell biological substances and on the cellular organelles such as amyloplasts, rather than on the cytoplasmic matrix where they are immersed as a consequence of their lower diamagnetic susceptibility. As a result, a nonuniform magnetic field exerts a ponderomotive force on the biological components in the opposite direction to the growing tip. This can result in intracellular magnetophoresis, and can account for inhibition of the root growth rate downwards. This inhibition would be stronger the lower the diamagnetic susceptibility.

Ultrastructural Aspects and Possible Origin of Cytoplasmic Channels Providing Intercellular Connection in Vegetative Tissues of Anthers1

Cytoplasmic channel represents a huge intercellular connection other than plasmodesma. They are proposed to play an essential role in controlling cell-to-cell trafficking of macromolecules. The present study ultrastructurally examined the occurrence, structure, and formation of this intercellular path in somatic tissues of wheat, tobacco, and onion anthers as well as their differences from those present in anther generative tissue. It was shown that cytoplasmic channels existed not only in the pollen mother cells, but also in both epidermis and vascular parenchyma of the anthers. In somatic tissues, they appeared as plasmallema-lined tubes 400–750 nm wide filled with nuclear or cytoplasmic material, the latter including cytoplasmic matrix, ribosomes, and filamentous structures. Their biogenesis seems to result from the thinning of the local portions of cell wall containing multiple plasmodesmata and the fusion of plasmodesmata in such regions induced by the wall-digesting enzymes released by nearby located vesicles. In contrast to the channels existing in the pollen mother cells of tobacco, the cytoplasmic channels in the epidermal or vascular parenchyma cells of wheat, onion, and tobacco anthers usually do not appear in groups, but are single. Perhaps this is the way for nuclear material to migrate from cell to cell via a single channel and then form a single chromatin spherical body in the adjoining cell. An individual cell could not accept the nuclear material from another cell while extruding its own to the third cell. Cell-to-cell migration of organelles through the cytoplasmic channels was not shown in the somatic tissues.

Secretion of acid phosphatase in Claviceps purpurea--an ultracytochemical study.

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.

Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells

Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells. Prolonged exposure of human peritoneal mesothelial cells (HPMC) to high glucose concentrations in peritoneal dialysate is the principal factor leading to matrix accumulation and thickening of the peritoneal membrane, accompanied by progressive deterioration of transport functions. These changes are mediated in part through protein kinase C (PKC) activation and the induction of transforming growth factor-beta 1 (TGF-beta 1). Emodin (3-methyl-1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells. How emodin modulates glucose-induced abnormalities in HPMC has not been elucidated and thus constitutes the theme of this study. We investigated the effects of emodin on the expression of PKC alpha, TGF-beta 1, fibronectin, and collagen type I in HPMC, and its effects on HPMC proliferation under physiologic (5 mmol) or high (30 mmol) glucose concentrations. Exposure of HPMC cultured with 5 mmol or 30 mmol D-glucose to emodin (20 microg/mL) resulted in an initial lag of proliferation by 2.3 to 2.7 days, but did not affect cell viability or morphology at confluence. D-glucose (30 mmol) induced TGF-beta 1 secretion in a time-dependent manner (3.72 +/- 0.29 and 4.30 +/- 0.50 pg/microg cellular protein at 24 hours and 48 hours respectively, compared to 2.13 +/- 0.23 and 2.65 +/- 0.32 pg/microg cellular protein at 24 hours and 48 hours, respectively for 5 mmol glucose; P < 0.001 at both time points). Such induction was ameliorated by emodin (20 microg/mL) (TGF-beta 1 concentration 2.25 +/- 0.15 and 2.96 +/- 0.33 pg/microg cellular protein at 24 hours and 48 hours, respectively, in the presence of emodin and 30 mmol D-glucose; P < 0.001 compared to 30 mmol D-glucose alone at both time points). Induction of TGF-beta 1 synthesis by 30 mmol D-glucose was associated with induction of PKC alpha, phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor-1 (ATF-1), and increased fibronectin and type I collagen translation. Emodin abrogated all these effects of concentrated glucose. Immunohistochemical staining showed that 30 mmol D-glucose induced cytoplasmic, perinuclear, and extracellular fibronectin and type I collagen expression by HPMC. Emodin reduced 30 mmol D-glucose-induced cytoplasmic and extracellular matrix synthesis to near basal levels. Our findings demonstrate that emodin ameliorates the undesirable effects of concentrated glucose on HPMC via suppression of PKC activation and CREB phosphorylation, and suggest that emodin may have a therapeutic potential in the prevention or treatment of glucose-induced structural and functional abnormalities in the peritoneal membrane.

Localization of brain-type fatty acid-binding protein in Kupffer cells of mice and its transient decrease in response to lipopolysaccharide.

Brain-type fatty acid-binding protein (B-FABP) was localized in Kupffer cells of liver of postnatal day 10 (P10) and older mice in immunolight and electron microscopy as well as by in situ hybridization histochemistry. The immunoreaction products were localized in the cytoplasmic matrix but not within the nucleus. After peritoneal injection of lipopolysaccharide (LPS), the immunoreaction for B-FABP decreased markedly in Kupffer cells at 1 h postinjection and thereafter gradually recovered to the preinjection level by 24 h postinjection, although no decrease in the mRNA expression was detected in Northern blotting throughout the course after the injection. The specific localization of B-FABP, but not the other FABPs, in Kupffer cells, and its rapid decrease after LPS injection suggest the intimate involvement of B-FABP in Kupffer cells in the inflammatory reaction, probably through mediation of n-3 polyunsaturated fatty acids, which are strong binders of B-FABP.

A novel retinol-binding protein in the retina of the swallowtail butterfly, Papilio xuthus.

Retinoid-binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol-binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol-binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand-binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplasmic matrix. All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes. Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.

Spatiotemporal changes of levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase, in subcellular compartments during spermatogenesis in the rat testis.

We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1). Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2). mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15-17; 3). nuclear labeling density suddenly increased in steps 12-14 to a maximum; 4). in cytoplasmic matrix, the density remained low in all steps; and 5). the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.