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D M Phillips

Publications and source records attributed to D M Phillips.

At least 91 records · Page 5Linked to original sources
PubMed1991

Microwave irradiation-accelerated in situ hybridization technique for HIV detection.

High frequency irradiation generated in a common household microwave oven was used to establish an in situ hybridization technique for rapid detection of HIV sequences in infected cells. A biotin-labeled DNA probe was subsequently detected either by an alkaline phosphatase-based colorimetric reaction or by fluorescence. When compared to standard hybridization procedures with radioactive or nonradioactive probes, microwave energy-mediated hybridization results in equal sensitivity and diminished background. The main advantage of this method, however, is the drastic reduction in time, allowing completion of the whole procedure, from sample preparation to hybrid signal visualization, within one hour. In addition to HIV detection, the approach described can be applied for the diagnosis of other viral infections and may stimulate the development of nucleic acid hybridization techniques based on microwave irradiation.

Biotin↗
PubMed1991

Assessment of the hygienic adequacy of a commercial hot boning process for beef by a temperature function integration technique.

The hygienic performance of a commercial hot boning process for beef carcasses was assessed by a temperature function integration technique. The potential proliferation of Escherichia coli was calculated from 50 temperature histories for the persistently warmest, microbially contaminated regions of product passing through both the carcass cutting and carton cooling phases of the process. The maximum calculated proliferation was similar to, but the average proliferation was more than, the respective values previously obtained for a beef side cooling process that complied with Good Manufacturing Practice. After upgrading of the carton cooling facility the process was re-assessed. Then, for a sample of 50 temperature histories, the maximum proliferation was less than, and the average proliferation was similar to, the respective values for the side cooling process. Observed proliferations of E. coli inocula in cooling cartons of product were compared with the proliferations calculated from temperature histories obtained from sites close to inocula. The pairs of calculated and observed values mostly agree within +/- 1 generation.

Animals↗
PubMed1991

Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir.

Ejaculated boar sperm were incubated with explants of porcine oviductal mucosa that had been dissected from the isthmic and ampullar regions of gilts. Sperm bound within minutes to the epithelial surfaces of the explants. Binding was not affected by region (isthmus or ampulla) nor day of estrous cycle (Day 0 or Day 10), but was increased by addition of 70 pg/ml 17 beta-estradiol to the medium. Scanning electron micrographs indicated that sperm bound, via the acrosomal region, to ciliated cells. After 24 h, the numbers of bound sperm dropped significantly, but the motility of the bound sperm did not. A mucous material that entrapped sperm was observed on the epithelial surfaces of 23/32 isthmic and only 4/32 ampullar explants. These results indicate that sperm sticking to ciliated cells and mucus can create a sperm reservoir in the isthmus, but the means by which sperm are released remain unknown.

Animals↗
PubMed1991

Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells.

Previous studies suggested that an extracellular steroid-binding protein, testosterone-estradiol-binding globulin (TeBG), can enter a variety of cells. Experiments were conducted to determine whether uptake of TeBG occurs by nonspecific fluid phase endocytosis or via a specific receptor-mediated process. In human breast carcinoma cells (MCF-7) maintained on serum-free medium, exposure to radiolabeled TeBG resulted in cellular uptake, which reached a plateau by 6 h and could be inhibited 80% by competition with unlabeled TeBG. Uptake was temperature dependent with cell-associated radioactivity at 37 C being 1.6-fold higher than at 4 C. Subsequent exposure of cells to pronase resulted in release of the cell-associated TeBG by 88% and 44% at 4 C and 37 C, respectively. After transfer to media devoid of TeBG, approximately 35% of cell-associated radioactivity was release into the medium at 37 C; it was not possible to distinguish whether this was released from the cell surface or from inside the cell. Investigation of the localization of TeBG-gold complexes by electron microscopy revealed that TeBG first binds to the plasmalemma. Within 15 min label appears in receptosomes, which fuse to form multivesicular endosomes. By 1 h all label is observed in multivesicular endosomes and lysosomes, most of which are in the Golgi zone. Localization of the internalized radioactivity using classical cell fractionation techniques showed it appears in a symmetrical band exhibiting the same buoyant density as the lysosomal marker acid phosphatase. The observations reported here show that: 1) TeBG binds to MCF-7 cells; 2) some of the bound TeBG is taken up via a mechanism with all the characteristics of receptor-mediated endocytosis; and 3) within these cells TeBG is localized in endosomes and lysosomes.

Breast Neoplasms↗
PubMed1991

Transmission of human immunodeficiency virus from monocytes to epithelia.

Physical contact between latent HIV-infected monocytes and epithelial cells appears to trigger rapid assembly and release of HIV into the enclosed space between donor and acceptor cells. During this process, monocytes form microvilli that intimately associate with epithelial membrane. HIVs sequestered in these sites are then internalized in the epithelial cells within phagocytic endosomes. Morphological evidence suggests that parts of virions fuse with the endosomal membrane and escape into the cytoplasm of the epithelium before endosome-lysosome fusion. In addition, virions in the lysosomal compartment are not necessarily degraded and may cause dissolution of phagolysosome membrane before HIV is damaged. This is reflected in the observation of free coated virions in the epithelial cytoplasm. Apart from the phagocytic mode of HIV entry, two other less common routes of HIV penetration were observed. One resembles direct HIV fusion with host membrane, which is similar to the entry mode of virus into CD4 lymphocytes. The second mode of entry resembles receptor-mediated endocytosis characterized by small pits and endosomes. Cell contact-mediated viral shedding and the diversity of HIV entry mechanisms are described for an in vitro model.

Cell Communication↗
PubMed1990

Identification of extracellular proteins in the rat cumulus oophorus.

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte-cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postovulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW approximately 81,000 and 100,000). The other two proteins were more basic and occurred at MW approximately 90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.

Animals↗
PubMed1990

Structure of the cumulus oophorus at the time of fertilization.

Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophorus of hamsters and rats. Observations on cumuli prepared in a variety of ways including different chemical fixation techniques and cryofixation freeze substitution were compared. It was concluded that the cumulus mucus is not arranged in lamellae or granules as has previously been suggested but is composed of molecules which form very fine filaments when properly fixed. Morphometric analysis of cumuli fixed either in situ or after being explanted into medium revealed that the distance between neighboring cumulus cells was greater with increasing distance from the oocyte. Morphometry revealed that, when placed into medium, the cumulus expands possibly due to hydration. Thus physiological experiments carried out on cumuli should be performed very shortly after cumuli are isolated. From their ultrastructure cumulus cells appear to be actively involved in protein synthesis and secretion as well as steroid production.

Animals↗
PubMed1990

Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro.

We have previously described an antigen (termed 2B1) on rat spermatozoa that is present on the plasma membrane overlying the tail domain. The antigen is mobile within the plane of the plasma membrane and a mAb to it blocks fertilization in vitro. In the present study we describe some dynamic properties of this antigen in relation to its topographical distribution. When spermatozoa were incubated in vitro in a capacitation medium and stained with 2B1 mAb/FITC-rabbit anti-mouse F(ab')2, strong fluorescence appeared over the acrosomal domain. Acute exposure of fresh spermatozoa to dissociating reagents (1 M NaCl or 5 mM 2-mercaptoethanol) or inducers of the acrosome reaction (lysolecithin + Ca2+ or A23187 + Ca2+) failed to mimic these effects. Spermatozoa prelabeled with FITC-2B1 IgG and then capacitated in the presence of excess "cold" 2B1 IgG also showed accumulation of fluorescence on the acrosomal domain, suggesting that the antigen had migrated from the tail. Migration was selective and Ca2(+)- and temperature-dependent but was not inhibited by metabolic poisons (NaF or NaN3). Motility was not obligatory for migration. Immunogold-labeling studies at the ultrastructural level showed that 2B1 antigen was restricted to the surface membrane over both the tail and the acrosomal domains and that during migration it did not change the type of membrane into which it was inserted. From a quantitative analysis of fluorescence on spermatozoa prelabeled with FITC-2B1 IgG and then capacitated, the amount of antigen that appeared on the acrosomal domain was approximately equivalent to that lost from the midpiece domain. The Mr of 2B1 antigen extracted from capacitated spermatozoa was 300-500 Da less than that extracted from noncapacitated cells, suggesting that the molecule had undergone processing concomitant with migration. These results are discussed in relation to mechanisms for targeting antigens to sites where they become physiologically active and are correctly positioned to participate in gamete recognition processes.

Animals↗
PubMed1990

Antisperm antibodies induce polyspermy by promoting adherence of human sperm to zona-free hamster eggs.

Certain antisperm antibodies (ASAs) in the sera of infertile men or women promote the penetration of zona-free hamster eggs by spermatozoa. We have shown previously that this enhancement of penetration occurs through mechanisms other than an alteration in the acrosomal structure of antibody-labelled spermatozoa. In the present study, small limited populations of antibody-labelled and antibody-free spermatozoa from fertile donors were observed serially by phase contrast, epifluorescence and scanning electron microscopy following their adherence to the oolemma of zona-free hamster eggs. The adherence of ASA-labelled spermatozoa to the zona-free hamster egg was markedly increased and occurred earlier when compared with antibody-free spermatozoa from the same ejaculate. The likelihood of entry of an antibody-labelled spermatozoon into the ooplasm, however, once adherent to the oolemma was not different to that of an antibody-free spermatozoa, and the ultrastructural steps of incorporation of antibody-labelled human spermatozoa were also similar to those observed during fertilization of hamster eggs by spermatozoa of several other species. These observations indicate that ASAs promote polyspermic fertilization of zona-free hamster eggs primarily through their enhancement of sperm--oolemmal binding.

Acrosome↗
PubMed1990

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype.

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

Actin Cytoskeleton↗
PubMed1990

Activin stimulates spermatogonial proliferation in germ-Sertoli cell cocultures from immature rat testis.

Activin and inhibin are peptide hormones produced in the gonads which may act as autocrine and/or paracrine regulators of testicular function. Sertoli cells produce inhibin, and it has recently been shown that Leydig cells can produce activin in vitro. To further explore the local actions of activin and inhibin in the testis, Sertoli and germ cells were isolated from immature rats and cocultured in vitro. In these cultures we demonstrate that activin A and activin B, but not inhibin A, stimulated spermatogonial proliferation in vitro. Activin increased [3H]thymidine incorporation 2- to 4-fold in cocultures after 48-72 h of treatment. Using autoradiography, the label was localized in the clusters of spermatogonia adhering to the Sertoli cell monolayer. Additionally, activin stimulated a reaggregation of the cultures into tubule-like structures. Fluorescence-activated cytometry was used to analyze the cell population based on size, DNA content, and lipid content. Sertoli cells were identified using Nile Red staining of intracellular lipid droplets; spermatogonia are Nile Red-negative. Activin treatment caused a marked increase in the fraction of Nile Red-negative cells in the cocultures. Activin also caused an increase in the percentage of these cells having 4C DNA. Lastly, specific binding of activin A to 2C, but not 4C, germ cells was demonstrated. These data demonstrate that activin acts as a regulator of spermatogonial proliferation in the male.

Activins↗
PubMed1990

Factors affecting the acrosome reaction in human spermatozoa.

Large pieces of human cumulus oophorus were exposed for 20-30 min to washed spermatozoa or to spermatozoa recovered after a swim-up procedure, and then fixed for electron microscopy. Spermatozoa of both populations penetrated deeply into the cumulus within that time, and none of 48 observed clearly had undergone an acrosome reaction (AR). As measured by fluorescence microscopy, an AR rate of 12% in spermatozoa obtained at 4 h following a swim-up increased to about 25% in samples incubated in culture dishes for approximately 20 h. However, this latter AR rate was no different in the presence or absence of a cumulus/oocyte complex, and was only moderately greater in 50% follicular fluid. Nor was it affected to any degree by the absence of calcium or by a low (26 degrees C) temperature, both of which are regulators of the physiological AR in other species. By contrast, a clear dose-related enhancement of the AR by the calcium ionophore A23187 was almost completely Ca2(+)-dependent. We conclude that the human cumulus oophorus does not rapidly induce an AR in spermatozoa capacitated in vitro and, unlike the situation in some other mammals, that washed human spermatozoa do not first require a period of capacitation in order to penetrate it. The results also point to the likelihood that ARs monitored in free-swimming human spermatozoa are for the most part spurious or artefactual, and they show that in-vitro AR rates in such populations do not parallel their fertilizing ability.

Acrosome↗
PubMed1990

Tracheal mucus clearance in high-frequency oscillation: effect of peak flow rate bias.

We have reported previously that high-frequency oscillation of the chest wall (HFO/CW) enhances the tracheal mucus clearance rate (TMCR) in dogs. This enhancement of TMCR may be due in part to the expiratory bias in peak flow rate (VE/VI greater than 1) that occurs during HFO/CW. We examined this factor in 8 anaesthetized, spontaneously breathing dogs by comparing TMCR during the following manoeuvers: 1) HFO/CW, applied by means of a thoracic cuff; 2) symmetric high-frequency oscillation via the airway opening (HFO/AO), applied by means of a piston pump driven by sinusoidal signal; 3) HFO/AO with an expiratory bias in peak flow, and 4) HFO/AO with an inspiratory bias in peak flow. All manoeuvers were of 5 min duration and were performed at 13 Hz and an oscillatory tidal volume of 1.5 ml.kg-1. In the latter two manoeuvers, the piston pump was driven by a nonsinusoidal signal such that peak VE/VI was greater than and less than unity, respectively. A high-impedance, cross-current flow of warmed, humidified air was provided at the tracheal tube. The order of manoeuvers 2, 3 and 4 was randomized, while manoeuver 1 was repeated at the end. TMCR was determined by direct bronchoscopic visualization of charcoal particle transport. Each HFO manoeuver was bracketed by a control period of spontaneous breathing. We found that TMCR during HFO/CW was 2.4 x control (p less than 0.001), in line with previous results.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Development of spermatozoa in the rhea.

We have examined the ultrastructural changes that take place during spermiogenesis in the rhea. Spermatozoa are characterized by a curved head and a midpiece. A thin rod extends from the anterior tip of the spermatozoon through the center of the nucleus. A 3-mu-long distal centriole occupies the entire midpiece. The principal piece is characterized by a small fibrous sheath and tiny dense fibers that are only observed in the region of the principal piece, which is immediately behind the annulus. During development a circular manchette surrounds the nucleus of young spermatids. Later the microtubules of the circular manchette become reorganized into a longitudinal manchette. A long distal and short proximal centriole are observed in early round spermatids. The distal centriole becomes associated with the plasma membrane. Later the proximal centriole is observed in association with the nucleus. The area around the centriole pair then accumulates dense material, which is associated with either the centrioles or the circular manchette. The longitudinal manchette forms and then disappears and mitochondria subsequently associate with the distal centriole. The long centriole of the rhea enables this species to develop a midpiece similar to the midpiece of mammalian sperm without the complex intercellular movements that characterize mammalian spermiogenesis.

Animals↗
PubMed1989

Status of the rat acrosome during sperm-zona pellucida interactions.

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluorescence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.

Acrosome↗
PubMed1989

Effects of anti-sperm antibodies on human sperm ultrastructure and function.

The ultrastructural appearance of a highly motile population of spermatozoa from a fertile donor was evaluated following antibody labeling with each of several sperm head-directed anti-sperm antibodies (ASAs) and subsequent incubation in a capacitating medium in vitro. Acrosomes were classified into categories which are believed to represent stages in the acrosome reaction. Quantitative analysis revealed no difference in acrosomal morphology of antibody-labeled sperm between treatment groups despite a varying degree of enhancement or inhibition of sperm egg-penetrating ability. We conclude that ASAs affect gamete interaction through mechanisms other than an alteration in the acrosome reaction.

Antibodies↗
PubMed1989

Assessment of temporal changes in pulmonary edema with NMR imaging.

Nuclear magnetic resonance imaging (NMRI) parameters [longitudinal relaxation time (T1), transverse relaxation time (T2), and signal intensity] acquired at a magnetic field of 2.35 T were validated with a study of nine different phantom gel solutions. This technique was then applied to study 13 anesthetized supine cats, among which 10 had lung edema induced by oleic acid (0.075 ml/kg); the result was compared with postmortem analyses of lung water. Three animals (series A) were imaged until the edema was first visualized in NMRI, usually 15-20 min after oleic acid infusion. Another seven animals (series B) were imaged over 4-5 h. As lung water increased, so did the signal intensity. When edema first appeared, T1, T2, and the volume of the edematous region within the slice in the upper lobes showed no gravity-dependent differences; this was confirmed by postmortem measurements (series A) of lung water. With time, gravity-dependent regions displayed greater volumes of edematous regions and greater T1 values (P less than 0.01), suggesting a continued accumulation of lung water. In comparison, nondependent regions displayed constant volumes of edematous region and lesser T1 values (P less than 0.01), suggesting an increased protein concentration but no change in lung water. This study suggests the potential applicability of NMRI parameters in the assessment of pulmonary edema.

Animals↗