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M W Hess

Publications and source records attributed to M W Hess.

At least 19 recordsLinked to original sources
PubMed2003

Of plants and other pets: practical aspects of freeze-substitution and resin embedding.

Representative tissues from higher plants (e.g. developing pollen, somatic anther tissues from the monocotyledonous angiosperm Ledebouria) and mammalian cell cultures were successfully cryoimmobilized by means of high-pressure freezing. Various substitution and embedding protocols were then evaluated considering the preservation of ultrastructural details, membrane staining, immunolabelling properties, as well as reproducibility and ease of use. Two types of recipe proved to be highly suitable for most applications, regardless of type, developmental stage or physiological conditions of the cells: (i) the best choice for morphology is still osmium in acetone (optionally supplemented with uranyl acetate) followed by embedding in Epon and/or Araldite; (ii) feasible approaches for immunocytochemistry are freeze-substitution with ethanol containing uranyl acetate and formaldehyde, or with pure acetone (in the case of fixation-sensitive antigens), followed by embedding with LR-white acrylic resin; though being far from optimal, these combinations represent, in my opinion, an acceptable compromise between labelling intensity, section stability, structural preservation and health hazards. Notably, the patterns observed in Ledebouria were consistent with data obtained from a broad range of other specimens from all kingdoms (e.g. leaves and callus cultures from angiosperms, gymnosperm roots with their ectomycorrhizal fungi, mammalian cell cultures and eubacteria). Finally, a warning is given as to the extractive potentials of embedding resins (Spurr's mixture, LR-white, but also Epon) being sometimes the cause of unacceptable artefacts, both in plant and in mammalian cells prepared by cryoimmobilization and freeze-substitution.

Animals↗
PubMed2001

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Adenosine Triphosphate↗
PubMed2001

Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy.

Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-micron-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation.

Blood Platelets↗
PubMed2000

Tracing uptake of C3dg-conjugated antigen into B cells via complement receptor type 2 (CR2, CD21).

Electron microscopy was used to study the internalization and delivery of ligands for complement receptor type 2 (CR2, CD21) to endocytic compartments of B-lymphoblastoid Raji cells. Opsonized antigen was mimicked with purified C3dg conjugated to colloidal gold. C3dg-gold bound specifically to the cell surface in a time-dependent manner, and preincubation of the cells with a monoclonal antibody blocking the CR2 ligand-binding site completely inhibited any C3dg-gold binding. Notably, the binding of C3d-gold was confined to cell surface protrusions, eg, microvilli. C3dg-gold was apparently internalized through coated pits located at the bases of microvilli and could be traced to different compartments of the endocytic pathway. The morphologic characteristics and intracellular distribution of these multivesicular or multilaminar structures were compatible with those of compartments known to harbor major histocompatibility complex (MHC) class II molecules. Immunolabeling showed that the internalized C3dg-gold colocalized with MHC class II in these structures. These data provide the first ultrastructural evidence that complement-coated antigens are endocytosed by antigen-nonspecific B cells by CR2 and are delivered to the compartments in which peptide loading for antigen presentation occurs. They support the notion that CR2 may play a role in antigen presentation by B cells regardless of B-cell receptor specificity. (Blood. 2000;95:2617-2623)

Antigen Presentation↗
PubMed2000

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

Animals↗
PubMed2000

Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Golgi apparatus.

High-pressure freezing and freeze-substitution were used to study Golgi ultrastructure and its brefeldin A-induced transformations in HepG2 human hepatoma cells. Cryoimmobilization arrested subcellular dynamics within milliseconds, thus considerably improving the temporal resolution in monitoring the very early effects of high brefeldin concentrations at the ultrastructural level (i.e., 20 microg/ml brefeldin applied for 35 s to 8 min). Moreover, this approach ruled out possible cumulative and/or synergistic effects of the drug and fixatives. Several findings differed from studies based on chemical fixation. In particular, Golgi breakdown did not proceed gradually but occurred in distinct steps. We found a conspicuous lag between the absence of nonclathrin coats on Golgi membranes after 30 s of brefeldin treatment and the disassembly of the stacks, which did not start until after 90 to 120 s. At this time, domains at the trans and cis faces separated from the stacks, starting tubulation and fragmentation. After 3-5 min the Golgi apparatus was completely replaced by loose meshworks of straight tubules of different sizes and staining properties; also frequent were bent tubules and vesicles forming glomerule-like structures. After 8 min all kinds of Golgi-derived structures had aggregated within huge clusters. The morphologically highly distinct structures found after brefeldin treatment could in part be correlated with particular Golgi domains in the control cells.

Brefeldin A↗
PubMed2000

Bactericidal activity of micromolar N-chlorotaurine: evidence for its antimicrobial function in the human defense system.

N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time. N-Chlorotaurine (12.5 to 50 microM) demonstrated a bactericidal effect i.e., a 2 to 4 log(10) reduction in viable counts, after incubation at 37 degrees C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5. 0), with a 3 to 4 log(10) reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated in N-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function of N-chlorotaurine and other chloramines during inflammation.

Adult↗
PubMed1999

A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.

Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction.

Adenosine Triphosphate↗
PubMed1998

Histological investigations on the Tyrolean Ice Man.

The 5,200-year-old Tyrolean Ice Man discovered in 1991 in the Otztal Alps is the world's most ancient known human glacier mummy. Histological investigation was aimed at 1) optimizing specimen preparation and 2) documenting the preservation state of (sub)cellular components. Minute pieces of frozen tissue were removed endoscopically from rib bone and cartilage, major blood vessels, oral cavity and alimentary tract, liver, spleen, diaphragm, respiratory system, femoral muscle and nerve, sympathetic trunk, brain, and skin. Double fixation with glutaraldehyde followed by osmium tetroxide and embedding in Epon/Araldite epoxy resins proved to be the method of choice for both light and transmission electron microscopy combined with classical histochemistry. In particular, mild evacuation of the desiccated tissue was determined to be essential to ensure homogeneous infiltration with fixatives and resins; as a result, sections of excellent quality could be obtained with any kind of sample. With regard to the preservation degree of (sub)cellular components, distinct tissue-specific patterns were observed. There were highly intact skeletal and connective tissues proper, however, most interestingly, there were remarkably intact nervous tissue components as well. By contrast, epithelial, muscle, and reticular connective tissues as well as blood had generally disintegrated due to autolysis, freeze/thaw damage, and adipocere formation. For a tentative interpretation of these patterns, we considered general aspects of cryopreservation, such as physicochemical properties of subcellular constituents and tissue physiology.

Adult↗
PubMed1997

Human prostatic smooth muscle cells in culture: estradiol enhances expression of smooth muscle cell-specific markers.

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 microM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle alpha-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs.

Actins↗
PubMed1996

Inhibition of submandibular and lacrimal gland infiltration in nonobese diabetic mice by transgenic expression of soluble TNF-receptor p55.

Besides a prominent mononuclear cell infiltration of the islets of Langerhans, nonobese diabetic (NOD) mice also show massive cellular infiltrates of the submandibular and lacrimal glands concomitant with histological signs of tissue damage. To obtain insights into the mechanisms operative during the initiation and progression of tissue damage, we followed by in situ hybridization the appearance of cells containing mRNA of the gene encoding the proinflammatory cytokine TNF-alpha in the cellular infiltrates. Cells expressing TNF-alpha are mainly located in infiltrates, are absent in nonaffected glands, and are preferentially found among CD4 T cells. Secretion of TNF-alpha by gland-infiltrating cells was confirmed by an ELISPOT procedure. Direct evidence for an instrumental role of TNF-alpha in initiation and progression of submandibular and lacrimal gland infiltration is provided by the observed significant reduction in the extent of infiltration in nonobese diabetic mice transgenic for a soluble TNF receptor p55 fused to the Fc part of human IgG3. This protection from infiltration is paralleled by decreased expression of the adhesion molecules ICAM-1 and VCAM-1 in submandibular and lacrimal glands. These data suggest a central role of TNF-alpha in the initiation and progression of autoimmune tissue destruction of salivary glands and indicate beneficial effects of soluble TNF receptors in the treatment of organ-specific autoimmune diseases.

Animals↗
PubMed1996

Differential in situ expression of the genes encoding the chemokines MCP-1 and RANTES in human inflammatory bowel disease.

Two chemotactic cytokines, monocyte chemoattractant protein-1 (MCP-1) and RANTES, possibly contribute to the recruitment and activation of leukocytes in inflamed tissues. The expression of these cytokine genes was evaluated in tissue sections from resected bowel segments of 14 patients with inflammatory bowel disease (IBD) and seven control patients by use of 35S-labelled antisense RNA probes. MCP-1 and RANTES transcripts were generally increased in the intestinal mucosa of patients with IBD, compared with controls. Whereas MCP-1 gene expression in the mucosa was restricted to the lamina propria, the gene coding for RANTES was expressed in intraepithelial lymphocytes and in the subepithelial lamina propria. Furthermore, MCP-1 mRNA, but not RANTES mRNA, was abundant in vessel-associated cells, such as endothelial cells, medial smooth muscle cells, and intraluminal cells; in smooth muscle cells of the intestinal tunica muscularis; and in cells of the myenteric plexus. Compared with controls, a significant increase of MCP-1-expressing cells was observed in tissue specimens from patients with IBD, in endothelial cells of venules, and in cells present in the lumen of intestinal vessels. Conversely, the expression of MCP-1 mRNA in smooth muscle cells and myenteric plexus cells appeared to be comparable in control and diseased intestines. The increased number of MCP-1 and RANTES mRNA-expressing cells in mucosa from patients with IBD suggests that these cytokines play a role in the pathogenesis of mucosal inflammation. Furthermore, the expression of the MCP-1 gene in vessel-associated cells may indicate its involvement in mechanisms regulating the adhesion of blood monocytes to endothelial cells.

Adult↗
PubMed1995

Immunocytochemical localisation of actin and profilin in the generative cell of angiosperm pollen: TEM studies on high-pressure frozen and freeze-substituted Ledebouria socialis Roth (Hyacinthaceae).

Actin was demonstrated for the first time at the EM level in the generative cell of mature angiosperm pollen by using immuno-gold labelling of high-pressure frozen and freeze-substituted Ledebouria socialis Roth anthers. In addition, profilin, an actin-monomer binding protein, is shown to coexist in the generative cell. We attribute the detection of actin and profilin to the applied cryomethods which yield a much better preservation of ultrastructure and antigenicity of delicate cytoskeletal constituents than conventional fixation techniques. Actin labelling was observed within the cytoplasm of the generative cell and became especially clear in close vicinity to microtubular bundles. Filamentous structures congruent with the actin labelling patterns do occur, but are not a frequent feature. Profilin was localised throughout the cytoplasm.

Actins↗
PubMed1993

The effect of cyclosporine treatment on the expression of genes encoding granzyme A and perforin in the infiltrate of mouse heart transplants.

Following activation of cytotoxic T cells and NK cells several genes encoding proteins putatively involved in cell-mediated cytotoxicity become expressed. The expression of genes encoding the cytotoxic T cell associated serine protease granzyme A and perforin was analyzed in cellular infiltrates of MHC mismatched (H-2d-->H-2k) heterotopic heart transplants both in immunosuppressed recipients treated with cyclosporine and in untreated recipients. Heart transplants were completely rejected by untreated animals on day 10 post-transplantation, whereas CsA treatment generally prolonged survival of the transplants beyond 30 days. In untreated recipients the number of granzyme A- and perforin-expressing cells in heart transplants increased from approximately 10 granzyme A-positive cells/mm2 and 1 perforin-positive cell/mm2 on day 2 posttransplantation to over 80 positive cells for both genes on day 5 posttransplantation. In contrast, these values remained always below 15 positive cells/mm2 for both genes between day 5 and day 30 posttransplantation in CsA-treated recipients. Comparison of the frequency of CD8+ T cells in the infiltrates showed that lower numbers of perforin and granzyme A-positive cells were mainly due to the immunosuppressive action of CsA rather than to reduced infiltration of transplants. The present study shows that expression of granzyme A and perforin gene can be used to discriminate between quiescent and activated cytotoxic cells also in immunosuppressed animals and further confirms that these can be used as sensitive markers for monitoring the fate of a transplant.

Animals↗
PubMed1993

A simple and inexpensive device for freeze substitution at 183 K/-90 C.

The freeze substitution device described here consists of commonly available materials, is easy to construct and to handle and provides maximal working safety. An adequate mass of copper and/or brass, serving as holder for 16 cryovials, is chilled to liquid nitrogen temperature. The vial holder is placed in a precooled thermos flask filled with ethanol and stored in an ultrafreezer at 193 K/-90 C. This ensures that the vial holder maintains temperatures of about 183 K/-90 C for more than 12 hr to minimize the risk of ice recrystallization.

Cryopreservation↗
PubMed1990

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

Animals↗