MorphoNeuroNet: an automated method for dense neurite network analysis.

High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases.

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins.

BACKGROUND: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression. METHODS: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses. RESULTS: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression. CONCLUSIONS: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics. GENERAL SIGNIFICANCE: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.

Repeated exposure of human fibroblasts to ionizing radiation reveals an adaptive response that is not mediated by interleukin-6 or TGF-ß.

Exposing cells to a low dose can protect them against a subsequent higher exposure. This phenomenon is known as adaptive response and is frequently observed in a variety of cells. Even though similarities are suspected with other non-targeted effects, such as bystander effects, the exact mechanism behind adaptive response is not fully clarified. In this study human primary fibroblasts were tested for their response to ionizing radiation (IR) after administrating a low priming dose (0.1-0.5Gy). Both the abundance of γH2AX as a marker for double-stranded breaks and the levels of cytokines, secreted in the medium, were monitored in time. Upon challenge, IR-primed cells showed modified γH2AX spot size distributions and altered repair kinetics, consistent with an adaptive response. In addition, 24h after priming with IR, four cytokines were significantly upregulated in the medium - GM-CSF (1.33×); IL6 (4.24×); IL8 (1.33×); TGF-ß (1.46×). In order to mimick the protective effect of IR priming, we primed the cells with either IL6 or TGF-ß. This did not elicit an altered γH2AX response as observed in IR-primed cells, indicating that the adaptive response in these primary fibroblasts is regulated in an IL-6 and TGF-ß independent manner.

A method for visualising polyelectrolyte distribution after polyelectrolyte conditioning of a biotic sludge.

Charge neutralisation is an important mechanism in (polyelectrolyte) conditioning of biotic sludges and required for efficient sludge dewatering. Based on results from streaming potential and zeta potential measurements, it has been suggested that charge neutralisation is more complete on the outside of the sludge flocs than on the inside. This paper discusses the development of a technique for assessing the spatial distribution of polyelectrolyte (PE) within sludge flocs. After flocculation with a fluorescently labelled PE, fluorescence microscopy can be used to visualise the distribution of the PE in the sludge flocs. Preliminary results indicate that the PE can penetrate relatively deep into the sludge flocs (and flocculi). Inhomogeneity in the PE distribution arises from differences in exposure to PE in different regions, and from differences in the affinity of the PE for different substances.

Medium-mediated DNA repair response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts.

Radiation induced bystander effects, either protective or adverse, have been identified in a variety of cells and for different endpoints. They are thought to arise from communication between cells through direct cell-cell contacts and via transmissible molecules secreted into the medium by targeted cells. We have investigated medium-mediated damage response in human dermal fibroblasts (HDF) after exposure to ionizing irradiation. We show that HDF experience an elevated level of double stranded DNA damage repair response when incubated with conditioned growth medium of irradiated cells. The magnitude of this response is much lower than observed for directly irradiated cells and is proportional to the radiation dose, as is its persistence across time. Since secretion of cytokines is one of the possible pathways linking targeted and non-targeted cells a multiplex analysis was performed. Four cytokines - IL6, IL8, MCP-1 and RANTES - were identified in the growth medium of irradiated cells after exposure to X-rays (2Gy). These cytokines were significantly upregulated and each cytokine showed differential upregulation kinetics. Finally we performed a functional analysis to see if IL6 and MCP-1 could induce gammaH2AX foci formation. IL6 caused a significant increase in spot occupancy compared to controls. Although only indicative MCP-1 appears to have the opposite effect as it caused a drop in spot occupancy. The combined addition of these two cytokines produced no significant response was observed. Both IL6 and MCP-1 have an effect on the gammaH2AX spot occupancy possibly linking these cytokines to the bystander response.

Systemic telomere length and preclinical atherosclerosis: the Asklepios Study.

AIMS: Peripheral blood leucocyte (PBL) telomere length (TL) is a systemic ageing biomarker and has been proposed to be an independent predictor of cardiovascular disease (CVD). We aimed at providing an explanation for this association by the evaluation of the biomarker value of PBL-TL in preclinical atherosclerosis. METHODS AND RESULTS: Peripheral blood leucocyte telomere length was assessed by telomere restriction fragment analysis in 2509 volunteers free from established CVD, aged approximately 35-55 years old, from the Asklepios Study cohort. Intima-media thickness (IMT) and plaque presence were determined by ultrasonography in both left and right carotid and femoral arteries. Peripheral blood leucocyte telomere length was not a significant independent determinant of IMT (P > 0.3) or plaque presence (P > 0.05), in either artery or either sex. In women but not in men, PBL-TL was a weak determinant of combined (carotid or femoral) plaque presence, adjusted for other risk factors (women: P = 0.03, men: P > 0.4). However, even in women presenting plaques, PBL-TL was still longer than in men. CONCLUSION: Since systemic TL is not a substantial underlying determinant of preclinical atherosclerosis, the association between CVD and TL cannot be explained by the fact that subjects with shorter inherited TL are predisposed to atherosclerosis.

High content analysis of human fibroblast cell cultures after exposure to space radiation.

Space travel imposes risks to human health, in large part by the increased radiation levels compared to those on Earth. To understand the effects of space radiation on humans, it is important to determine the underlying cellular mechanisms. While general dosimetry describes average radiation levels accurately, it says little about the actual physiological impact and does not provide biological information about individual cellular events. In addition, there is no information about the nature and magnitude of a systemic response through extra- and intercellular communication. To assess the stress response in human fibroblasts that were sent into space with the Foton-M3 mission, we have developed a pluralistic setup to measure DNA damage and inflammation response by combining global and local dosimetry, image cytometry and multiplex array technology, thereby maximizing the scientific output. We were able to demonstrate a significant increase in DNA double-strand breaks, determined by a twofold increase of the gamma-H2AX signal at the level of the single cell and a threefold up-regulation of the soluble signal proteins CCL5, IL-6, IL-8, beta-2 microglobulin and EN-RAGE, which are key players in the process of inflammation, in the growth medium.

Telomere length and cardiovascular risk factors in a middle-aged population free of overt cardiovascular disease.

Evidence assembled over the last decade shows that average telomere length (TL) acts as a biomarker for biological aging and cardiovascular disease (CVD) in particular. Although essential for a more profound understanding of the underlying mechanisms, little reference information is available on TL. We therefore sought to provide baseline TL information and assess the association of prevalent CVD risk factors with TL in subjects free of overt CVD within a small age range. We measured mean telomere restriction fragment length of peripheral blood leukocytes in a large, representative Asklepios study cohort of 2509 community-dwelling, Caucasian female and male volunteers aged approximately 35-55 years and free of overt CVD. We found a manifest age-dependent telomere attrition, at a significantly faster rate in men as compared to women. No significant associations were established with classical CVD risk factors such as cholesterol status and blood pressure, yet shorter TL was associated with increased levels of several inflammation and oxidative stress markers. Importantly, shorter telomere length was associated with an increasingly unhealthy lifestyle, particularly in men. All findings were age and gender adjusted where appropriate. With these cross-sectional results we show that TL of peripheral blood leukocytes primarily reflects the burden of increased oxidative stress and inflammation, whether or not determined by an increasingly unhealthy lifestyle, while the association with classical CVD risk factors is limited. This further clarifies the added value of TL as a biomarker for biological aging and might improve our understanding of how TL is associated with CVD.

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria.

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.

Methylglyoxal suppresses TNF-alpha-induced NF-kappaB activation by inhibiting NF-kappaB DNA-binding.

Methylglyoxal is a cytotoxic metabolite that is produced in vivo mainly from glycolysis. Increased production of methylglyoxal can be induced by tumor necrosis factor and occurs in a number of pathological conditions, including diabetes and neurodegenerative disorders. Methylglyoxal is highly reactive and can modify proteins, which results in the formation of advanced glycation end products. Yet, we, and others, have recently proposed a role for methylglyoxal as a signaling molecule. In this study, we show that methylglyoxal inhibits TNF-induced NF-kappaB activation and NF-kappaB-dependent reporter gene expression by inhibiting the DNA binding capacity of NF-kappaB p65. Methylglyoxal slightly delayed, but did not inhibit, TNF-induced degradation of IkappaBalpha and strongly inhibited TNF-induced NF-kappaB-dependent re-synthesis of IkappaBalpha. The TNF-induced nuclear translocation of NF-kappaB p65 was also delayed, but not inhibited, in the presence of methylglyoxal. TNF-induced phosphorylation of p65 was not affected by methylglyoxal. We show that the conserved Cys 38 residue, which is located in the DNA binding loop of NF-kappaB p65 and responsible for the redox regulation of the transcription factor, is involved in the methylglyoxal-mediated inhibition of p65 DNA-binding. Furthermore, overexpression of p65 inhibited TNF-induced cell death; however, in the presence of exogenously added methylglyoxal, overexpression of p65 caused far greater TNF-induced cell death. These findings suggest that methylglyoxal provides another control mechanism for modulating the expression of NF-kappaB-responsive genes and that methylglyoxal may be responsible for tipping the balance towards TNF-induced cell death in cells with constitutive NF-kappaB activation.

Telomere shortening is associated with malformation in p53-deficient mice after irradiation during specific stages of development.

The natural ends of linear chromosomes, the telomeres, recruit specific proteins in the formation of protective caps that preserve the integrity of the genome. Unprotected chromosomes induce DNA damage checkpoint cascades and ultimately lead to senescence both in mouse and man in a p53 dependent manner and initial telomere length setting therefore determines the proliferative capacity of each cell. Yet, only little information is available on telomere biology during embryonic development. We have previously shown that the p53 gene plays a crucial role in the development of malformations (exencephaly, gastroschisis, polydactyly, cleft palate and dwarfism) in control and irradiated mouse embryos. Here, we investigated telomere biology and the outcome of radiation exposure in wild type (p53+/+) and p53-mutant (p53+/-- and--/--) C57BL mouse foetuses irradiated at three different developmental stages. We show that telomeres are significantly shorter in malformed foetuses as compared to normal counterparts. In addition, our results indicate that the observed telomere attrition is primarily associated with p53-deficiency but is also modulated by irradiation, more specifically during the gastrulation and organogenesis stages. In conclusion, we formulate a hypothesis in which telomere shortening is linked to the absence of p53 in mouse foetuses and that when, in the presence of shorter telomeres, these foetuses are irradiated, the chance for the occurrence of developmental defects increases substantially.

Genotoxicity in wood mice (Apodemus sylvaticus) along a pollution gradient: exposure-, age-, and gender-related effects.

We investigated the effects of environmental pollution on genetic damage in wood mice (Apodemus sylvaticus) by means of the comet assay, with special attention to the role of age and gender as potential confounding variables. The present study was carried out at four sites along a pollution gradient in the vicinity of Antwerp (Belgium), with a nonferrous smelter as the main pollution source. We measured the concentration of heavy metals (Cd, Co, Cr, Cu, Fe, Mn, Pb, and Zn) in mouse liver and kidney and the concentration of organochlorine compounds (polychlorinated biphenyls and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) in mouse muscle tissue to assess individual exposure. Cadmium exposure was very high at the sites closest to the smelter, and exposure to this metal decreased with increasing distance from the smelter. Exposure to the other pollutants was low to moderate at the different sites. Genetic damage was higher in mice from populations in the vicinity of the nonferrous smelter compared with that in the control populations. A significant increase in genetic damage with age was observed at the most polluted sites, but not at the control sites. Genetic damage was higher in male mice than in female mice at the most polluted site, but not at the other areas. Yet, no obvious relationship was found between individual pollutant levels and individual genetic damage levels. We conclude that the comet assay can be used to compare genotoxicity at the population level if the confounding variables of gender and age are taken into account. However, its use for individual health risk assessment remains questionable.

Combined Exposure to Simulated Microgravity and Acute or Chronic Radiation Reduces Neuronal Network Integrity and Survival.

During orbital or interplanetary space flights, astronauts are exposed to cosmic radiations and microgravity. However, most earth-based studies on the potential health risks of space conditions have investigated the effects of these two conditions separately. This study aimed at assessing the combined effect of radiation exposure and microgravity on neuronal morphology and survival in vitro. In particular, we investigated the effects of simulated microgravity after acute (X-rays) or during chronic (Californium-252) exposure to ionizing radiation using mouse mature neuron cultures. Acute exposure to low (0.1 Gy) doses of X-rays caused a delay in neurite outgrowth and a reduction in soma size, while only the high dose impaired neuronal survival. Of interest, the strongest effect on neuronal morphology and survival was evident in cells exposed to microgravity and in particular in cells exposed to both microgravity and radiation. Removal of neurons from simulated microgravity for a period of 24 h was not sufficient to recover neurite length, whereas the soma size showed a clear re-adaptation to normal ground conditions. Genome-wide gene expression analysis confirmed a modulation of genes involved in neurite extension, cell survival and synaptic communication, suggesting that these changes might be responsible for the observed morphological effects. In general, the observed synergistic changes in neuronal network integrity and cell survival induced by simulated space conditions might help to better evaluate the astronaut's health risks and underline the importance of investigating the central nervous system and long-term cognition during and after a space flight.

Telomere attrition as ageing biomarker.

Telomeres, the tandem-repeated hexamers at the termini of mammalian chromosomes, form protective complexes in association with specific proteins that together with telomerase, a specialised telomere-synthesizing enzyme, regulate telomere length. Telomere shortening is associated with cellular senescence and is implicated in tumorigenesis and cancer. Hence, mean telomere length has emerged as a replicative clock within each population of cells and the tissues and organs they build up in vitro and, consequently, as a biomarker for biological ageing in vivo. Chronological ageing per se does not parallel biological ageing, yet accurate and reliable biomarkers are lacking to distinguish between them. The question remains as to whether telomere dynamics is a determinant or merely a predictor of human biological age over and above chronological ageing. Although several reports have suggested a link between telomere attrition and ageing phenotypes and disorders, both reference values and a complete set of determinants are missing. Within this review, current evidence and knowledge on telomere length and telomere erosion rates reported, are summarised.

Comparison of different protocols for telomere length estimation by combination of quantitative fluorescence in situ hybridization (Q-FISH) and flow cytometry in human cancer cell lines.

BACKGROUND: The end of eukaryotic chromosomes terminates with nucleoprotein structures called telomeres. They insure several functions including capping the end of the chromosomes, ensuring their stability and protecting them from end-to-end fusion and preventing the activation of the DNA damage checkpoints. MATERIALS AND METHODS: A flow-FISH methodology, i.e. quantitative fluorescence in situ hybridization (Q-FISH) in combination with flow cytometry, has been developed in our laboratory in order to estimate telomere length in three human cancer cell lines: K-562 (chronic myelogenous leukaemia), IM-9 (multiple myeloma) and 1301 (T cell lymphoblastic leukaemia). Telomeres were visualised after hybridisation with FITC-labelled PNA (Peptide Nucleic Acid) probes. We evaluated the most critical steps of the flow-FISH protocol to ensure reproducibility. Different methodological set ups were compared. Three fixation procedures (ethanol 80%, methanol 80% and formaldehyde 4%) were tested besides different fixation times (15 min and 60 min) as well as hybridization times (2 h and overnight). For each of these protocols the following parameters were compared: forward scatter (related to the cell size), side scatter (related to the cell granularity), DNA (FL3 and FL4 fluorescence) and PNA content (FL1 fluorescence) using an EPICS XL flow cytometer. RESULTS: Regarding the fixation procedures, methanol proved to be the best, followed by ethanol and formaldehyde, with respect to the efficiency to measure the different parameters cited above. Indeed, fixation using methanol gave the optimal PNA signal compared to using ethanol and formaldehyde in two of the studied cell lines (K-562 and 1301); the difference observed was highly significant in the 1301 cell line. The duration of fixation did not show significant interference in the reproducibility of the results for the three cell lines studied. An overnight hybridization appeared to be more effective when compared to the 2-h hybridization in the case of the K-562 cell line. CONCLUSION: The most important steps of the flow-FISH technique, namely the fixative procedure, as well as the hybridization and the fixation times, were investigated. Considering the latter, suitable protocols were set up for routine and fast telomere length estimation in the cancer cell lines.

Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana.

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.

Simultaneous in situ profiling of DNA lesion endpoints based on image cytometry and a single cell database approach.

Analyzing the integrity of DNA is one of the most frequent used endpoints for risk assessment of chemical and physical agents. In the framework of a radiobiological space experiment, this work aimed at having (1) a histochemical tool for the in situ assessment of DNA damage in as long as 20 days old fixed cell cultures, (2) a comprehensive tool for the quantification of different types of DNA lesions, and (3) a methodology of sampling thousands of nuclei based on confocal microscopy, automated stage scanning and digital image processing. For this purpose several fixatives and permeabilization techniques were tested together with the combinatorial use of terminal dUTP transferase-mediated nick end-labeling (TUNEL) and the DNA polymerase I mediated in situ nick translation. These biochemical tools are useful for scoring DNA single and double breaks, and oxidative lesions. Ltk(-) cells were exposed either to hydrogen peroxide or heavy ion beam irradiation. Combination of paraformaldehyde fixation, sodium citrate permeabilization and heat gave the best staining results. A three-channel fluorescence methodology was established including a DNA counter stain for nucleus identification and normalization of DNA content. Communication between confocal imaging software, image analysis software and a relational database proved to be pivotal for a semi-automated high-end single cell analysis and storage of images. In this way, DNA damage data per nucleus can be traced back to the original image. As much as 2500 cells could be analyzed in situ within a day and correlations drawn between different DNA lesion endpoints.

Neurofibromin is actively transported to the nucleus.

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors 'shuttle' between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28-49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.

Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells.

Milk protein-derived peptides with angiotensin-converting enzyme (ACE) inhibitory activity can reduce blood pressure in hypertensive subjects. The lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) is an ACE-inhibitory peptide released by tryptic digestion from the milk protein beta-lactoglobulin. Its ACE-inhibitory activity is 100 times lower than that of captopril. The latter is known to inhibit the release of the vasoconstrictor endothelin-1 (ET-1) by endothelial cells. The effects of ALPMHIR on the endothelium are currently unknown. In this study, the influence of ALPMHIR on release of ET-1 by endothelial cells was investigated. The basal ET-1 release of the cells was reduced by 29% (p<0.01) in the presence of 1 mM ALPMHIR, compared to 42% (p<0.01) for 0.1 mM captopril. Addition of 10 U/ml thrombin to the incubation medium increased the release of ET-1 by 66% (p<0.01). Co-incubation of 10 U/ml thrombin with 1 microM captopril or with 0.1 mM ALPMHIR inhibited the stimulated ET-1 release by 45% (p<0.01) and by 32% (p<0.01), respectively. These data indicate that dietary peptides, such as ALPMHIR, can modulate ET-1 release by endothelial cells. These effects, among other mechanisms, may play a role in the anti-hypertensive effect of milk protein-derived peptides.

Dynamic changes in microtubular cytoskeleton of human postmature oocytes revert after ooplasm transfer.

OBJECTIVE: Study of the influence of ooplasm transfer on the microtubule dynamics in human postmature oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. MATERIALS(S): Human in vitro matured (IVM) oocytes (n = 65). Experimental groups: In set 1, sibling oocytes were processed either within 2-3 hours ("young"; n = 16) or at 12-14 hours after maturation ("presumably postmature," or PPM; n = 14). In set 2, young and PPM oocytes (n = 6 and 10, respectively) were assigned to be ooplasm donors and recipients, respectively. In set 3, PPM oocytes were used as ooplasm donors (n = 2) and recipients (n = 4). Control groups: Metaphase II oocytes from superovulated golden hamsters in set 1; sibling oocytes of ooplasm donor young (n = 4) and PPM oocytes (n = 7) in set 2; and sibling PPM oocytes in set 3 (n = 2). INTERVENTION(S): Immunocytochemistry for alphatubulin with or without treatment with taxol (Paclitaxel, a microtubule-enhancing agent) in set 1; aspiration and microinjection of approximately 20 picolitres ooplasm from donor young and PPM oocytes into recipient PPM oocytes in sets 2 and 3, respectively. Taxol treatment and tubulin immunocytochemistry on ooplasm recipients and control young and PPM sibling oocytes. MAIN OUTCOME MEASURE(S): Morphology and pattern of the microtubules in the spindle and ooplasm as evaluated by confocal microscopy and three-dimensional image reconstructions. RESULT(S): In set 1, taxol-untreated young oocytes had normal spindle morphology and orientation to the oolemma with no microtubules in the ooplasm. Taxol-treated young oocytes revealed markedly broadened spindle poles and minimal or absent ooplasmic microtubules. Taxol-untreated PPM oocytes had variable spindle morphology and a notable increase in cortical ooplasmic microtubules. Taxol treatment of PPM oocytes resulted in a marked increase in ooplasmic microtubules in addition to a broadening of spindle poles and formation of polar asters. In set 2, control young and PPM oocytes had the same findings as the corresponding oocytes in set 1. However, all ooplasm recipient PPM oocytes showed a striking diminution in ooplasmic microtubules, despite the taxol treatment, compared with their sibling PPM control oocytes in set 2 and PPM ooplasm-injected PPM oocytes in set 3. CONCLUSION(S): Postmature oocytes exhibit a dynamic increase in ooplasmic microtubules. However, these changes revert after transfer of ooplasm from young oocytes.