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PubMed1983

An unusual cylindrical body in interstitial cells of rat testis.

Testes of 6 rats were processed routinely for electron microscopy and, as an incidental finding, interstitial (Leydig) cells were found to contain bundles of unusual cylindrical bodies or macrotubules. These cytoplasmic structures were found in testes of only 2 of the 6 rats and varied in number from 2 to 346 per cell profile, usually in parallel array but with an irregular orientation in approximately 5% of the cells. The macrotubules were up to 11 microns long, showed a diameter of 130 nm with a wall of 18-20 nm and an inside diameter of about 94 nm. The wall appeared to be formed by two membranes 6-7 nm thick showing a unit membrane structure with a central electron-lucent space of 6 nm. In several instances, the wall membranes appeared corrugated, which may account for the apparent formation of the wall by a spiralling small tubule or tubules of 18 nm diameter. Several macrotubules showed continuity between their wall membranes and elements of endoplasmic reticulum and occasionally two adjacent macrotubules showed continuity at their ends in a U-form. Similar if not identical structures have been described previously in four studies of interstitial cells of the rat renal medulla, in one instance correlated with water-deprivation. They have been considered an alteration in the endoplasmic reticulum. Also, they have been reported in pig uterine glands in pregnancy. They may represent a cellular response to an unrecognized physiopathological state.

Animals↗
PubMed1980

Micromegakaryocytes in Human Bone Marrow.

Micromegakaryocytes (MMK) were defined morphologically by the cell area, nucleus form and cytoplasmic structure. Bone marrow smears of 7,156 patients were retrospectively analyzed. MMK were found most frequently and abundantly in acute non-lymphatic leukaemia, chronic myeloid leukaemia and pre-leukaemia. The presence of more than 10% MMK in the megakaryocyte population suggest a pre-leukaemic condition or non-lymphatic leukaemia. The platelet production of MMK is probably quantitatively normal although a functional defect is suspected.

Acute Disease↗
PubMed1984

Effects of Lonidamine on murine and human tumor cells in vitro. A morphological and biochemical study.

Lonidamine induces in murine and human tumor cells severe morphological damage of the mitochondria and other cytoplasmic structures both 'in vitro' and 'in vivo'. Biochemical studies have demonstrated that the drug decreases oxygen consumption and lactate production. The sensitivity of human tumor cells is not related to their histotype. Lonidamine's effects on mitochondria, glycolysis, pentose phosphate pathway, and aromatase activity are discussed.

Animals↗
PubMed1997

Ultrastructural mucosal appearance in the ileal neobladder.

METHODS: 15 patients with ileal neobladder underwent endoscopic biopsy at different postoperative intervals. The specimens were analyzed by electron microscopy in order to evaluate the evolution of the mucosal changes ultrastructurally. RESULTS: No significant change was observed 3 months after the operation. After 6 months, the number and height of the microvilli were reduced, the cell borders crooked and the terminal web upset. After 12 months disappearance of the glycocalyx, increased lysosomal features, increased activity of the muciparous cells, rounded mitochondria and loss of the polarized disposition of the cytoplasmic organelles were detected in the enterocytes. We observed no other substantial change after 24 months and more. CONCLUSIONS: Progressive modifications occur in the cytoplasmic structures involved in the absorptive process. They do not seem to begin before 3 months and are almost totally completed after 1 year.

Aged↗
PubMed1997

Characterization of acidic vesicles in multidrug-resistant and sensitive cancer cells by acridine orange staining and confocal microspectrofluorometry.

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange. Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO. In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles. The resulting rise in the dye's oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry. In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 +/- 10%) than the value (48 +/- 8%) from K562-sensitive cells (p < 0.001). This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 microM), or nigericine (0.3 microM) all suppressed the initial difference of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one. The same effects were induced by 50 microM verapamil, a pleiotropic drug-resistance modulator. Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles.

Acridine Orange↗
PubMedDate not supplied

Formation of porcine hepatocyte spheroids for use in a bioartificial liver.

Xenogeneic hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, from multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 microns. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Urea-genesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.

Animals↗
PubMed2002

Hepatic lipidosis in pregnant captive American bison (Bison bison).

Hepatic lipidosis, a hallmark lesion of lipid mobilization disorders in ruminants, was noted in four 3-year-old, pregnant bison (Bison bison) after periods of anorexia that progressed to recumbency and death. The affected bison were part of a herd at the National Animal Disease Center (NADC) that was used for brucellosis vaccine research. Microscopically, the liver contained swollen hepatocytes with numerous, variably sized, round, smoothly contoured vacuoles that displaced cytoplasmic structures. Hepatocytes in all zones of the lobule were affected equally. Hypoglycemia, decreased total carbon dioxide, elevated gamma-glutamyltransferase, elevated alkaline phosphatase, and increased nonesterified fatty acid levels were noted. As in the case of cattle, altered nutritional demands of late gestation combined with management factors such as obesity, nutrition, stress, and concomitant disease may be critical in the pathophysiology of lipid mobilization disorders in bison. Additionally, stressors unique to this research herd likely contributed to fatal hepatic lipidosis.

Animals↗
PubMed1988

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes.

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.

Animals↗
PubMed1996

Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry.

We used fracture-label and label-fracture cytochemistry in conjunction with the phospholipase A2-colloidal gold (PLA2-CG) technique to study the distribution of phospholipids in ejaculated boar spermatozoa. These techniques provide visualization of the topographical distribution of phospholipids in freeze-fractured sperm membranes in a three-dimensional view. In various freeze-fractured boar sperm membranes and crossfractured cytoplasmic structures, quantitative analysis revealed that the nuclear envelope membranes and the nuclear content possessed the highest labeling density of PLA2-CG. Moderate labeling was detected over acrosomal membranes, especially the inner acrosomal membrane. Replicas of both protoplasmic and exoplasmic fracture faces of the plasma membrane of boar sperm head showed a relatively low density of PLA2-CG labeling. Moreover, a differential distribution of phospholipids was seen over the protoplasmic face of the plasma membrane domains of the sperm head, which showed the highest concentration of gold particles in the postacrosomal region, followed by the equatorial segment and the anterior acrosome region. The PLA2-CG labeling densities over the post-acrosomal region and the equatorial segment were significantly higher than that over the anterior acrosome region. In the flagellum, an intense labeling was also seen over crossfractured mitochondria, dense fibers, and fibrous sheath. The protoplasmic fracture face of the plasma membrane over the middle piece, the annulus, and the principal piece was moderately labeled by PLA2-CG. No significant difference in mean labeling density of PLA2-CG was detected among the three membrane domains. In label-fracture preparations, exoplasmic halves of the plasma membrane of the head and the middle piece of the tail were uniformly labeled with PLA2-CG. However, the annulus and principal piece were devoid of PLA2-CG binding sites. These results indicate that differential distribution of phospholipids associated with the boar sperm membranes may reflect phospholipid composition of membrane domains characteristic of special physiological functions.

Animals↗
PubMed2004

Interactions between the megakaryocyte/platelet-specific beta1 tubulin and the secretory leukocyte protease inhibitor SLPI suggest a role for regulated proteolysis in platelet functions.

Platelet-restricted beta1 tubulin is required for optimal thrombopoiesis and discoid cell shape. To identify interacting factors, we used the divergent beta1-tubulin C-terminus as the bait in a yeast 2-hybrid screen of megakaryocyte (MK) cDNAs. We isolated secretory leukocyte protease inhibitor (SLPI), a serine protease antagonist characterized principally as a secreted factor with multiple roles in inflammation. SLPI is expressed in MKs and platelets in 2 discrete compartments. One pool resides in punctate cytoplasmic structures, whereas a significant fraction localizes along peripheral microtubules (MTs) and is lost with cold-induced MT disruption or in beta1 tubulin(-/-) platelets. These findings reveal unexpected interaction between a prominent cytoskeletal protein and an inhibitor of proteolysis. SLPI(-/-) mice show intact proplatelet formation, platelet numbers and shape, and marginal MT bands; thus, SLPI is not essential for thrombopoiesis. However, SLPI is released upon platelet activation, which also reverses its association with the resting marginal band. Platelet SLPI inhibits neutrophil elastase, an activity that is reduced when beta1 tubulin is absent. We conclude that SLPI localizes in part along the MK and platelet MT cytoskeleton by virtue of specific interactions with beta1 tubulin. SLPI may thus have unanticipated roles in MK and platelet functions, including regulated proteolysis after activation.

Animals↗
PubMed2004

Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex.

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Adaptor Protein Complex 3↗
PubMed2003

HER-2-targeted antisense oligonucleotide results in sensitization of head and neck cancer cells to chemotherapeutic agents.

Existing HER-2 targeted therapies for human head and neck cancers, usually administered in combination with chemotherapeutic drugs or irradiation, include monoclonal antibodies to HER-2, receptor tyrosine kinase inhibitors and HER-2 specific immunotoxins. Instead of targeting the existing protein, interference with HER-2 mRNA translation by antisense oligonucleotides may be a more efficient method to downregulate levels of HER-2 protein for combination therapy. To test this hypothesis we have used a phosphorothioate pentadecamer, complementary to the HER-2 mRNA initiation codon region (AS HER-2 ODN), to increase sensitivity to four chemotherapeutic agents in human head and neck cancer cell lines, all of which express low levels of the HER-2 protein. To improve delivery into tumor cells, the AS HER-2 ODN was complexed with our previously established folate-liposome delivery system. Cell survival assays and Western blot analysis data demonstrated that folate-liposome mediated AS HER-2 oligonucleotide treatment inhibited cell growth and HER-2 expression, and induced apoptosis in SCC-25CP cells. Moreover, there was a synergistic effect on the percent of apoptotic cells. Additionally, the combination of folate-liposome-AS HER-2 ODN and CDDP had a synergistic effect on the induction of apoptosis. Using confocal microscopy, FITC labeled ODN (FITC-ODN) in complex with folate-liganded, rhodamine (Rh) labeled, cationic liposomes was observed to enter SCC-25CP head and neck tumor cells within 3 to 6 h. Intracellularly, the FITC-ODN separated from the Rh-folate-liposomes, and FITC-ODN accumulated in the nucleus while Rh-liposomes remained in punctate cytoplasmic structures. Thus, folate-liposome-mediated delivery of AS HER-2 ODN has potential as a new means of increasing the responsiveness of head and neck cancer to conventional chemotherapy.

Antineoplastic Agents↗
PubMed1988

Induction of adenosine 3',5'-monophosphate-dependent protein kinase subunits during adipogenesis in vitro.

Fatty acid metabolism in adipocytes is known to be regulated by the intracellular transducer cAMP. This study was undertaken to determine the temporal and hormonal regulation of cAMP-dependent protein kinase during the differentiation of preadipocyte mesenchymal cells to adipocytes. For this we have used a stable cell line (TA1) in which the undifferentiated preadipocyte acquires adipocyte functions and morphology after growth to confluence. We observed that synthesis of type I and II cAMP-dependent protein kinases was induced during the adipogenic conversion of growth-arrested TA1 cells. In preconfluent cells, neither mRNAs encoding regulatory subunits (RI, RII beta) and catalytic subunit (C alpha) nor the peptides themselves were detectable. Within several days of growth arrest at high cell density, mRNAs for RI, RII beta, and C alpha were detectable in total RNA extracted from cell populations. The subunits themselves were detectable in some, but not all, of the cells by indirect immunofluorescence. Immunoblotting of cytosolic extracts indicated that RI and the beta-isoform of RII (mol wt = 52,000) were expressed in these cells. Analysis of subunit presence or absence in single cells by immunofluorescence also indicated that kinase subunit expression preceded the accumulation of lipid droplets within the cells. Further, the subunits were predominantly associated with a reticular cytoplasmic structure (Golgi apparatus?) abutting the nucleus. Conversion of TA1 cells to adipocytes can be accelerated by indomethacin (125 microM) or dexamethasone (1 microM) treatment, compounds that also enhanced the accumulation of RII beta and C alpha mRNAs. Within 2-3 days of addition of indomethacin to confluent cultures, RII beta message content is increased about 20-fold, and protein content is increased about 5-fold relative to those in untreated cultures. C alpha mRNA content is increased about 5-fold relative to that in untreated cells. The response to dexamethasone requires 6-7 days, and changes in RII beta message levels were the most pronounced. We also observed the induction of mRNAs for the functionally relevant mRNA lipoprotein lipase in indomethacin-treated cells. In addition to this apparent transcriptional regulation of kinase subunit expression, we provide evidence for regulation at the posttranscriptional level. Within a differentiated culture, there exist stem cells that can be selected, will repopulate the dish, and will again differentiate into adipocytes upon growth arrest at high cell density. In preconfluent populations of these stems cells, unlike the preconfluent TA1 cells originally plated, both RII beta and C alpha messages were present.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Regulation of the mitotic and meiotic cell cycles in the male germ line.

Mammalian gametogenesis provides a unique system in which to study cell-cycle regulation. Furthermore, understanding the genetic program controlling the mitotic and meiotic divisions of the germ line will provide insight into understanding infertility and new directions for contraception. Male and female germ cells have stages of cell-cycle regulation in common, including a mitotic proliferative stage, entry into meiosis, completion of a reductive division, and entry into a quiescent state awaiting signals at fertilization. However, the timing of these events - and, indeed, even the stage of development at which these events occurs - differs in the two sexes. The genes involved in controlling these specialized mitotic and meiotic cycles of mammalian germ cell differentiation are only now being identified. They include a complex array of kinases, phosphatases, regulatory proteins (e.g., cyclins), and an equally complex array of substrates, including components of the nuclear and cytoplasmic structures involved in cell division. This chapter provides an overview of our current understanding of cell-cycle regulation in mammalian mitotic cells and the importance of restriction points. A summary of observations regarding the expression of various cell-cycle regulatory genes in mouse gametes is provided, along with comments on interesting differences between mitotic and meiotic cells. Finally, the role of the novel A-type cyclin, cyclin A1, during male meiosis is discussed in depth.

Animals↗
PubMed1994

The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins and functions in cyst formation.

Oogenesis in Drosophila takes place within germline cysts that support polarized transport through ring canals interconnecting their 15 nurse cells and single oocyte. Developing cystocytes are spanned by a large cytoplasmic structure known as the fusome that has been postulated to help form ring canals and determine the pattern of nurse cell-oocyte interconnections. We identified the adducin-like hts product and alpha-spectrin as molecular components of fusomes, discovered a related structure in germline stem cells and documented regular associations between fusomes and cystocyte centrosomes. hts mutations completely eliminated fusomes, causing abnormal cysts containing a reduced number of cells to form. Our results imply that Drosophila fusomes are required for ovarian cyst formation and suggest that membrane skeletal proteins regulate cystocyte divisions.

Animals↗
PubMed1997

A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.

Germline stem cells play a pivotal role in gametogenesis; yet little is known about how they are formed, how they divide to self-renew, and how these processes are genetically controlled. Here we describe the self-renewing asymmetric division of germline stem cells in the Drosophila ovarian germline, as marked by the spectrosome, a cytoplasmic structure rich in membrane skeletal proteins. The ontogeny of the spectrosome marks the lineage of germline stem cells. We identified two new groups of mutations in which the divisional asymmetry is disrupted. The first, which we refer to as ovarette (ovt) mutations, was shown to correspond to a novel class of mutations in the pumilio locus. Since pumilio is known to posttranscriptionally repress the expression of target genes at earlier stages of germ cell development, our results suggest that a similar activity is needed to maintain germ line stem cells. We have also identified a second and novel gene, piwi, whose mutations abolish germline stem cell division.

Animals↗
PubMed1992

Expression of wild-type and nuclear localization-deficient human lamin A in chick myogenic cells.

Previous analysis of chick embryonic muscle (CEM) differentiation in vivo and in ovo demonstrated that lamin A accumulation to steady-state levels preceded the accumulation of muscle-specific proteins. These observations have suggested the appearance of A-type lamins may be important for differentiation. To test this hypothesis, we have temporally and quantitatively altered the expression of A-type lamins in CEM cells by transient transfection of wild-type (wt; pHLA) or nuclear localization-deficient (NLd; pHLA-del) human lamin A expression plasmids. Transfected CEM cells synthesized the wt and NLd human lamin As to high levels, both of which were resistant to high-salt extraction. The wt human lamin A localized to the nucleus, whereas the NLd protein showed cytoplasmic staining patterns, as well as time-dependent nuclear localization. The presence of endogenous chicken lamins A and B2 in NLd human lamin A cytoplasmic structures suggested the interspecies lamin copolymerization. Thus, this approach may provide a possible method for analysis of lamin-lamin or lamin-lamina component interactions in vivo. With regard to muscle differentiation, CEM cells transfected with either pHLA or pHLA-del demonstrated moderate and transient increased levels of the muscle-specific myosin heavy chain and creatine kinase activity. These increases appeared temporally and quantitatively to reflect the transient accumulation of the human lamin As. In contrast, beta-tubulin and skeletal tropomyosin protein accumulations appeared unaffected. On the basis of these results, we suggest that nuclear lamina content and structure may play a limited, permissive and indirect role in the temporally regulated expression of the myogenic phenotype.

Animals↗
PubMed1994

Molecular characterization of two functional domains of CLIP-170 in vivo.

CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected antibodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal microtubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.

Amino Acid Sequence↗