PubMed Health⌕ Search

Biomedical subjects

P Van Oostveldt

Publications and source records attributed to P Van Oostveldt.

At least 19 recordsLinked to original sources
PubMed2006

Two novel techniques to detect follicles in human ovarian cortical tissue.

BACKGROUND: Ovarian tissue cryopreservation and transplantation are becoming increasingly important issues for preserving female fertility as shown by recent successes in restoring ovarian activity and even fertility. Primordial follicle content before transplantation is a key issue for success. We investigated two novel methods to detect primordial follicles in human ovarian cortical tissue strips. METHODS: The first method used the fluorescent mitochondrial stain rhodamine 123 in combination with laser scanning confocal microscopy (LSCM). The first method used the fluorescent mitochondrial stain rhodamine 123 (R123) in combination with laser scanning confocal microscopy (LSCM). The second used a simple stereomicroscopic method with glass-bottom dishes for detecting primordial follicles in ovarian cortical tissue strips. Potential toxic effects of R123 and of the exposure to confocal laser were investigated in a mouse ovarian allograft model. RESULTS: Follicles were visible as white spots in thin cortical strips using LSCM in single and fast scanning at low magnification, allowing a fair estimation of the number of primordial follicles present. Using the second method, ovarian follicles were also visible using glass-bottom dishes under the stereomicroscope, although tissue thickness and density were limiting factors of its success. DISCUSSION: Follicles can be visualized in human cortical ovarian strips with R123 in combination with LSCM. Stereomicroscopy using glass-bottom dishes and transmitted illumination is a simple alternative method and has the advantage of allowing further safe clinical use of the analysed tissue.

Adult↗
PubMed2005

Telomere length versus hormonal and bone mineral status in healthy elderly men.

Telomeres, the termini of linear chromosomes, exert a key role in the process of cellular ageing. Progressive telomere shortening is implicated in senescence in vitro and ample evidence exists to support the hypothesis that telomere length is correlated with chronological age and ageing phenotypes in vivo. In this study, we assessed whether mean telomere length of peripheral blood leukocytes predicts age-associated bone loss and/or is related to sex steroid status in an elderly healthy male population (71-86 years). Out of this population, we selected 110 samples for telomere restriction fragment (TRF) length analysis. Fasting blood was analysed for testosterone, estradiol, sex hormone binding globulin and biochemical markers of bone turnover. Also, the bioavailable fractions of sex steroids were calculated. Bone mineral density was measured at baseline and longitudinal follow-up was available for 84 men. We found that mean TRF length was inversely correlated with age (r=-0.19; P=0.049). Although no correlations were found with sex steroids or BMD at baseline, age corrected mean TRF length was associated with longitudinal bone loss for different distal forearm sites (P<0.05). Further studies are required to confirm our results, yet in this study, the predictive value of telomere length for bone loss appears to be substantial, hence underscoring the role of telomere length as a biomarker of ageing phenotypes.

Aged↗
PubMed2005

Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

Animals↗
PubMed2005

Inhibition of bovine sperm-zona binding by bovine herpesvirus-1.

The purpose of the present study was to identify a potential interference of bovine herpesvirus-1 (BoHV-1) with sperm-oocyte interactions during bovine in vitro fertilization. An inhibition of almost 70% of sperm-zona binding was observed when bovine cumulus-denuded oocytes were inseminated in the presence of 10(7) 50% tissue culture infective dose/ml BoHV-1. The inhibitory effect of BoHV-1 on sperm-zona binding was mediated by an interaction of the virus with spermatozoa, but not with oocytes. Treatment of spermatozoa with BoHV-1, however, did not affect sperm motility and acrosomal status. Antiserum against BoHV-1 prevented the virus-induced inhibition of sperm-zona binding, indicating that BoHV-1 itself affects the fertilization process. In order to investigate which BoHV-1 glycoprotein(s) are responsible for the virus-sperm interaction, BoHV-1 was treated with monoclonal antibodies against the viral glycoproteins gB, gC, gD and gH prior to insemination. Anti-gC completely prevented the inhibitory effect of BoHV-1 on sperm-zona binding, while anti-gD caused a reduction of this inhibition. Further evidence for the involvement of gC and gD in the virus-sperm interaction was provided by the fact that purified gC and gD decreased sperm-zona binding in a dose-dependent way with gC being more effective than gD. These results indicated that BoHV-1 inhibits bovine sperm-zona binding by interacting with spermatozoa. The binding of BoHV-1 to a spermatozoon is mediated by the viral glycoproteins gC and gD, and therefore seems to be comparable with the mechanisms of BoHV-1 attachment to its natural host cell.

Acrosome Reaction↗
PubMed2003

Four-dimensional imaging and computer-assisted track analysis of nuclear migration in root hairs of Arabidopsis thaliana.

Nuclear migration is a fundamental mechanism necessary for the proper growth and development of many eukaryotic organisms. In this study root hairs of Arabidopsis thaliana were used as a research model to gain insight into the dynamics of nuclear migration. Root hairs are long tubular outgrowths of epidermal cells and are responsible for the uptake of water and nutrients. During the development of root hairs, the nucleus migrates into the hair after the bulge is formed. The position of the nucleus relative to the tip plays an essential role in the growth process. However, what is happening to the nucleus in full-grown root hairs is still unclear. To study nuclear dynamics in living root hair cells, stably transformed plants with the fusion proteins Histone2B-YFP and NLS-GFP-GUS were used. Four-dimensional confocal laser scanning microscopy made it possible to monitor the exact position of the nucleus in different root hairs. To analyse the sequential positions of the nuclei in the root hairs, a new computer-assisted method was developed. After track analysis a number of parameters could be extracted from the movies, such as the average speed, the amplitude, direction factor and the range of movement in the root hairs. Our results show that nuclei do not reach a final position in full-grown root hairs and this sustained movement seems to be more similar in root hairs lying close to each other. Moreover, with this methodology it could be quantitatively demonstrated that the integrity of actin is necessary for nuclear movement.

Arabidopsis↗
PubMed2002

Comparing telomere length of sister chromatids in human lymphocytes using three-dimensional confocal microscopy.

BACKGROUND: The length of the terminal sequences of linear chromosomes changes dynamically during cellular proliferation. A crucial element in the study of telomere-related regulation mechanisms is the ability to measure telomere lengths of individual chromosomes. Individual telomere lengths can be measured using digital imaging fluorescence microscopy-based techniques. We extended this method using confocal microscopy for the acquisition of three-dimensional (3D) images. Consequently, variations in measured signal intensities due to erroneous focusing are avoided. METHODS: We employed our 3D telomere sizing method to compare telomere lengths of sister chromatids within metaphase preparations from human lymphocytes. The samples were treated following a quantitative fluorescence in situ hybridization (Q-FISH) protocol using fluorescein isothiocyanate (FITC)-labeled telomeric peptidic nucleic acid (PNA) probes and propidium iodide (PI) counterstain. RESULTS: We demonstrated that the telomere lengths of two sister chromatids are not necessarily equal in human lymphocytes. Profound statistical analysis demonstrated significant differences in the distribution of the sister chromatid telomere lengths, but we were not able to prove a discrete distribution of telomere sister ratios. These telomere length differences were more apparent in older individuals. CONCLUSION: Whereas the majority of sister telomere pairs have equal lengths, surprisingly, a minority was significantly different in each individual studied. We are convinced that these observations are not linked to the methodology or the protocol applied. We suggest that a biological phenomenon might be involved.

Adult↗
PubMed2002

Inositol 1,4,5-trisphosphate receptor function in human oocytes: calcium responses and oocyte activation-related phenomena induced by photolytic release of InsP(3) are blocked by a specific antibody to the type I receptor.

Type I inositol 1,4,5-trisphosphate-sensitive receptors (InsP(3)R) are expressed in human oocytes and may be involved in operating the Ca(2+) release triggered by the fertilizing sperm. This study examines the contribution of type I InsP(3)R in operating Ca(2+) release in human oocytes secondary to InsP(3) itself, using a specific function-blocking antibody in conjunction with photolytic release of microinjected InsP(3). Intracellular Ca(2+) responses were assessed in oocytes microinjected with only caged InsP(3) in experiment set A, while in experiment sets B and C, sibling oocytes were injected with caged InsP(3) and the blocking antibody or a corresponding volume of medium, prior to flash photolysis. In experiment set C, certain fertilization-related phenomena (cortical granule exocytosis and chromatin configurations) were assessed using optical sections and three-dimensional image reconstructions obtained from a confocal laser scanning microscope. In experiment set A, photolytic release of InsP(3) triggered a Ca(2+) response (increase from approximately 100 to 220 nmol/l followed by an exponential recovery, n = 8) and a wave in the oocytes that spread from the stimulation point to the opposite pole. In set B, photolytic InsP(3) release generated Ca(2+) responses in control oocytes (n = 9), but not in the antibody-injected oocytes (n = 7). In set C, cortical granule exocytosis and anaphase chromosome configurations were noted in the control oocytes after flash photolysis (n = 6). These changes were completely absent in antibody injected oocytes as their cortical granules were intact and the chromosomes were in metaphase. These oocytes had also lacked Ca(2+) responses as in set B (n = 5). This study demonstrates the functional presence of type I InsP(3)R-operated Ca(2+) channels in human oocytes and further suggests an active role of InsP(3) in triggering the Ca(2+) rise and secondary activation phenomena at fertilization.

Antibodies, Monoclonal↗
PubMed2001

Involvement of cellular cytoskeleton components in antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and this may interfere with antibody-dependent cell lysis. We investigated the role of actin, microtubules, clathrin, and dynein, the major cellular components involved in physiological endocytosis during this virological internalization. Porcine monocytes were infected in vitro for 13 h and afterward treated with different concentrations of colchicine, cytochalasin D, latrunculin B, and amantadine-HCl, which inhibit polymerization of microtubules, actin/clathrin, actin, and clathrin, respectively. This resulted in a significant reduction of internalization compared to the nontreated control, indicating that these components are involved in the process. A double labeling was performed during the internalization process and a clear colocalization of actin, microtubules, clathrin, and dynein with the viral glycoproteins was observed at different stages during the internalization process. We conclude that these cellular components are used by PrV to generate the antibody-induced internalization of viral glycoproteins.

Actins↗
PubMed2001

Non destructive analysis of the wax layer of apple (Malus domestica Borkh.) by means of confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was used to non-destructively analyse the changes in the structure and thickness of the cuticle during storage of apples (Malus domestica Borkh.). Interpretation of the confocal images was performed by comparison with scanning electron microscopy and environmental scanning electron microscopy images. The natural reflectance of the wax and the auto-fluorescence of the underlying cells made it possible with CLSM to distinguish the wax from the underlying layers without any pretreatment of the fruit. The thickness of the consecutive layers (wax, cutin, cells) could be estimated from measurements of the reflection and fluorescence intensities as a function of the number of pixels. The mean wax-layer thickness measured in this way amounted to 2.58 microm, 3.41 microm or 4.14 microm for the cultivars Jonagold, Jonagored and Elstar, respectively. Changes in the wax structure and cells of the same important Belgian apple cultivars as mentioned above were monitored during nine months of storage in ultra low oxygen and after exposure to ambient conditions. The changes in the wax ultrastructure and cell morphology are likely related to water losses and specific protection of the apple cultivars against water losses during storage and shelf life.

Fruit↗
PubMed2000

Role of anti-gB and -gD antibodies in antibody-induced endocytosis of viral and cellular cell surface glycoproteins expressed on pseudorabies virus-infected monocytes.

The addition of porcine pseudorabies virus (PrV)-specific polyclonal IgG antibodies to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and major histocompatibility complex (MHC) class I. Using PrV deletion strains, it was shown that gB and gD are essential for the process to occur. The purpose of the current study was to evaluate whether antibodies directed against single viral glycoproteins are able to induce endocytosis. It was shown that monoclonal antibodies directed against viral glycoprotein gB and gD, but not against gC and gE, are able to induce internalization of their respective ligand. Adding a combination of monoclonal antibodies against gB and gD resulted in endocytosis levels, comparable to the endocytosis levels observed when adding porcine PrV-specific polyclonal antibodies. The addition of genistein and tyrphostin 25, two inhibitors of tyrosine kinase activity, abolished endocytosis induced by monoclonal anti-gB and -gD antibodies in a concentration-dependent manner. The addition of similar concentrations of tyrphostin 1, an inactive tyrphostin, had no effect on endocytosis. It was also shown that a mixture of polyclonal, but not monoclonal, antibodies against gB and gD is able to induce cointernalization of MHC class I. This indicates that MHC class I cointernalization results from a passive catching of the molecules rather than from a specific interaction of the MHC class I molecules with one or more viral glycoproteins. In conclusion, it can be stated that antibody-induced crosslinking of gB and gD induces the activation of a tyrosine phosphorylation-dependent signal transduction pathway, leading to their endocytosis. Cointernalization of other viral glycoproteins and MHC class I is most likely caused by a passive catching of these molecules in the gB and gD aggregates.

Animals↗
PubMed2000

Colocalization of dynactin subunits P150Glued and P50 with melanosomes in normal human melanocytes.

Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous reports we showed the presence of MT-associated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Dermatol 2000;143:258-306). We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. Therefore, in this study, we investigated the in vitro expression of dynactin subunits P150Glued and P50 in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription-polymerase chain reaction and northern blot analysis. In an attempt to gain an insight into the subcellular localization of dynactin, immunofluorescence and immunoelectron microscopy (IEM) studies were performed. The two isoforms of P150Glued and P50 are expressed in all studied skin cells. Immunofluorescence staining shows punctate distributions for P150Glued and P50 in melanocytes. P150Glued shows a clear centrosomal staining and accentuation in the dendrite tips. P50 is also accentuated in the perinuclear area and dendrite tips. Immunofluorescence double-labeling with a melanosome marker showed apparent colocalization of both P150Glued and P50 with melanosomes. By IEM, P50 is detected on the surface of the majority of melanosomes in melanocytes. The colocalization of different subunits of the dynactin complex with melanosomes is consistent with the earlier finding of cytoplasmic dynein association with melanosomes and supports the hypothesis that this complex could form a link between cytoplasmic dynein and the melanosomal membrane.

Blotting, Northern↗
PubMed2000

Cytoplasmic dynein colocalizes with melanosomes in normal human melanocytes.

BACKGROUND: Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. OBJECTIVES: We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. METHODS: Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. RESULTS: The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. CONCLUSIONS: The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.

Adult↗
PubMed2000

In this issue

Explore the source record for details and available documents.

Journal Article↗
PubMed1999

Single cell and tissue specific methods for evaluation of radiation and microgravity effects.

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.

Animals↗
PubMed1999

Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity.

OBJECTIVE: To study the effect of delayed intracytoplasmic sperm injection (ICSI) on the fertilization and cleavage of human in vitro matured (IVM) oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): The experimental group consisted of 73 spare germinal vesicle-stage oocytes from 25 patients. The control group consisted of sibling in vivo matured oocytes from the same patients that were subjected to ICSI in the clinical program. INTERVENTION(S): Equal numbers of sibling IVM oocytes were subjected to ICSI either soon after maturation (30 hours, group 1) or after a 6-hour delay (36 hours, group 2). In a subsequent set of experiments, spermatozoa were labeled with a fluorescent mitochondria-specific vital dye and injected into 17 IVM oocytes that were intentionally aged by 6-8 hours. The resultant zygotes were fixed. MAIN OUTCOME MEASURE(S): Incidence of fertilization and cleavage, numbers and mean diameters of pronuclei, and incidence of zygotes with significant pronucleus size asynchrony. Identification of the male pronucleus by its proximity to the fluorescent sperm midpiece remnant. RESULT(S): Group 2 had significantly lower rates of normal fertilization (60%) than the control group (82.9%) and significantly lower cleavage rates (46.7%) than both group 1 (85%) and the control group (98.1%). The incidence of oocytes that developed one pronucleus and pronucleus size asynchrony was significantly higher in group 2 (32% and 40%, respectively) than in group 1 (4% and 5%, respectively) and in the control group (4.1% and 4.4%, respectively). All the zygotes with significant pronucleus size asynchrony that developed after delayed ICSI with labeled spermatozoa showed proximity of the fluorescent sperm midpiece remnant to the smaller pronucleus. CONCLUSION(S): For IVM oocytes, the incidence of one pronucleus, pronucleus size asynchrony (possibly related to a smaller male pronucleus), and cleavage failure increase when ICSI is delayed after maturation. Thus, the timing of ICSI is critical for optimum fertilization of IVM oocytes.

Cell Nucleus↗
PubMed1999

Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research.

This review introduces the basics of fluorescence recovery after photobleaching (FRAP) from a theoretical and an instrumentational approach. The most interesting and innovative applications with a pharmaceutical point of view are briefly discussed and possible future applications are suggested. These future applications include research on the mobility of macromolecular drugs in macro- or microscopic pharmaceutical dosage forms, mobility, and binding of antitumor drugs in tumor tissue, intracellular trafficking of gene complexes and mobility of drugs in membranes prior to transmembrane penetration. The paper is also intended to be an introductory guideline to those who would like to get involved in FRAP related experimental techniques. Therefore, comprehensive details on different setups and data analysis are given, as well as a brief outline of the problems that may be encountered when performing FRAP. Overall, this review shows the great potential of FRAP in pharmaceutical research. This is complemented by our own results illustrating the possibility of performing FRAP in microscopic dosage forms (microspheres) using a high resolution variant of FRAP.

Cell Membrane↗