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PubMed1972

The metachronal wave of lateral cilia of Mytilus edulis.

The form of beat of cilia and the structure of the metachronal wave on the lateral gill epithelium of Mytulus edulis have been studied on living material by interference-contrast microscopy and stroboscopic illumination, and compared with the same features in rapid-fixed preparations studied by light microscopy and with the scanning electron microscope. The most striking finding is that the beat of the cilia is not planar, as previously assumed, but involves a sideways movement in the recovery stroke Previous reports on nonplanar ciliary beating from protozoan examples describe a planar effective stroke and a counterclockwise rotation in the recovery stroke; in this molluscan example there is a clockwise rotation in the recovery stroke The lateral inclination of the cilia in the recovery stroke is in the same direction as the propagation of the waves, and the orientation of cilia in the recovery stroke is thought to determine whether the waves move to the left or right of the direction of the effective stroke

Animals↗
PubMed1975

Studies of intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes (PMNS). I. Role of contact inhibition of locomotion.

Intercellular invasion is the active migration of cells on one type into the interiors of tissues composed of cells of dissimilar cell types. Contact paralysis of locomotion is the cessation of forward extension of the pseudopods of a cell as a result of its collision with another cell. One hypothesis to account for intercellular invasion proposes that a necessary condition for a cell type to be invasive to a given host tissue is that it lack contact paralysis of locomotion during collision with cells of that host tissue. The hypothesis has been tested using rabbit peritoneal neutrophil granulocytes (PMNs) as the invasive cell type and chick embryo fibroblasts as the host tissue. In organ culture, PMNs rapidly invade aggregates of fibroblasts. The behavior of the pseudopods of PMNs during collision with fibroblasts was analyzed for contact paralysis by a study of time-lapse films of cells in mixed monolayer culture. In monolayer culture, PMNs show little sign of paralysis of the pseudopods upon collision with fibroblasts and thus conform in their behavior to that predicted by the hypothesis.

Animals↗
PubMed1982

Reactivation of intestinal epithelial cell brush border motility: ATP-dependent contraction via a terminal web contractile ring.

Various models have been put forward suggesting ways in which brush borders from intestinal epithelial cells may be motile. Experiments documenting putative brush border motility have been performed on isolated brush borders and have generated models suggesting microvillar retraction or microvillar rootlet interactions. The reported Ca++ ATP-induced retraction of microvilli has been shown, instead, to be microvillar dissolution in response to Ca++ and not active brush border motility. I report here studies on the reactivation of motility in intact sheets of isolated intestinal epithelium. Whole epithelial sheets were glycerinated, which leaves the brush border and intercellular junctions intact, and then treated with ATP, PPi, ITP, ADP, GTP, or delta S-ATP. Analysis by video enhanced differential interference-contrast microscopy and thin-section transmission electron microscopy reveals contractions in the terminal web region causing microvilli to be fanned apart in response to ATP and delta S-ATP but not in response to ADP, PPi, ITP, or GTP. Electron microscopy reveals that the contractions occur at the level of the intermediate junction in a circumferential constriction which can pull cells completely apart. This constriction occurs in a location occupied by an actin-containing circumferential band of filaments, as demonstrated by S-1 binding, which completely encircles the terminal web at the level of the intermediate junction. Upon contraction, this band becomes denser and thicker. Since myosin, alpha-actinin and tropomyosin, in addition to actin, have been localized to this region of the terminal web, it is proposed that the intestinal epithelial cell can be motile via a circumferential terminal web contractile ring analogous to the contractile ring of dividing cells.

Adenosine Triphosphate↗
PubMed1984

Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment in Rous sarcoma virus-transformed rat cells.

Regions associated with cell-substratum contact or attachment in Rous sarcoma virus (RSV)-transformed rat fibroblasts (RR1022 cells) were identified by reflection-interference microscopy. Electron microscopy of such regions revealed the presence of discrete membrane-associated structures composed of a paracrystalline lattice of hexagons and pentagons to which actin filaments appear to be attached. Staining of actin by biotin-labeled heavy meromyosin showed that transformed cells, unlike normal fibroblasts, lack prominent actin fibers, and that, instead, much of the fluorescence is concentrated in loci corresponding to locations of transient association between the cell and the substratum. In stationary cells, such loci were found in rosette formation, predominantly in the region beneath the nucleus. In cells engaged in active movement, such as during migration into a wound, the actin-containing spots were concentrated in the region of the leading edge. A similar pattern of staining was observed with antibody to gelsolin, a 91,000-dalton Ca2+-dependent actin filament-shortening protein. Since the action of gelsolin on actin is reversible and dependent on physiologically relevant changes in calcium concentration, the localization of gelsolin, together with actin-bundling proteins such as alpha-actinin, in the regions containing many small microfilament bundles on the ventral side of cytoplasm suggests that gelsolin may be a component of the mechanism for the disassembly and assembly of actin during the dissolution and reformation of structures for cell-substratum contact during cell locomotion. Regulation of gelsolin activity was not dependent on protein phosphorylation, as shown by lack of 32P-incorporation into gelsolin in either transformed or normal fibroblasts.

Actins↗
PubMed1975

The interaction in vitro between human polymorphonuclear leukocytes and Neisseria gonorrhoeae cultivated in the chick embryo.

Cultivation of Neisseria gonorrhoeae in the allantoic cavity of 10-day chick embryos ensured the following necessary properties for subsequent quantitive in vitro phagocytosis studies of viable gonococci: log phase of growth, resistance to the cidal effect of fresh human serum, maintenance of colonial type, and absence of clumping. Employing a modification of the Maaloe technique, phagocytosis of log-phase type 1 and 2 gonococci by human PMN leukocytes did not occur in the presence or absence of serum. These findings indicate that log-phase type 1 and 2 gonococci possess antiphagocytic surface factors Stationary-phase organisms of the same colonial type were ingested and rapidly killed by human PMN leukocytes under similar experimental conditions, thus emphasizing the necessity to employ log-phase gonococci in the study of phagocytosis and antiphagocytic surface factors. Log-phase type 4 gonococci were ingested and rapidly killed by human PMN leukocytes in the presence of fresh human serum but not heat-inactivated serum or in the absence of serum. Morphologic studies demonstrated that log-phase viable gonococci attach to the surface membrane of human PMN leukocytes. Interiorization of avirulent but not virulent organisms was observed in the presence of fresh human serum. Gonococci-human PMN leukocyte interactions thus provide a model for the investigation of the nonimmunologic and immunologic parameters associated with the attachment and ingestion stages of phagocytosis.

Animals↗
PubMed2004

The collagen structure of bovine intervertebral disc studied using polarization-sensitive optical coherence tomography.

Polarization-sensitive optical coherence tomography (PS-OCT) is used to measure the birefringence properties of bovine intervertebral disc and equine flexor tendon. For equine tendon the birefringence delta n is (6.0 +/- 0.2) x 10(-3) at a wavelength of 1.3 microm. This is somewhat larger than the values reported for bovine tendon. The surface region of the annulus fibrosus of a freshly excised intact bovine intervertebral disc displays an identical value of birefringence, delta n = (6.0 +/- 0.6) x 10(-3) at 1.3 microm. The nucleus pulposus does not display birefringence, the measured apparent value of delta n = (0.39 +/- 0.01) x 10(-3) being indistinguishable from the effects of depolarization due to multiple scattering. A clear difference is found between the depth-resolved retardance of equine tendon and that of bovine intervertebral disc. This apparently relates to the lamellar structure of the latter tissue, in which the collagen fibre orientation alternates between successive lamellae. A semi-empirical model based on Jones calculus shows that the measurements are in reasonable agreement with previous optical and x-ray data. These results imply that PS-OCT could be a useful tool to study collagen organization within the intervertebral disc in vitro and possibly in vivo and its variation with applied load and disease.

Animals↗
PubMed2002

Spindle observation in living mammalian oocytes with the polarization microscope and its practical use.

The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. Conventional methods to image spindles rely on fixation and transmission electron microscope or immunofluorescence staining and fluorescence microscope, so they provide limited value to studies of spindle dynamics and human clinical in vitro fertilization. A new orientation-independent polarized light microscope, the LC Polscope, was used to examine the bi-refringent spindles in living mammalian oocytes. It was found that spindles could be imaged with the Polscope in living oocytes in all mammals so far examined, including hamster, mouse, cattle, human, and rat. The first polar body did not accurately predict the spindle location in most metaphase II oocytes. Intracytoplasmic sperm injection (ICSI) could be performed by monitoring spindle position. Studies in humans indicated that, aftr ICSI, higher fertilization and embryonic developmental rates could be achieved in oocytes with than without bi-refringent spindles. Because spindles in most mammalian oocytes are extremely sensitive to slight changes in temperature, maintenance of temperature at 37 degrees C is crucial for normal spindle function. As chromosomes#10; are usually associated with microtubule fibers in the spindles, the position of chromosomes could be indirectly located by imaging spindles. Removing spindles under the Polscope can achieve an enucleation#10; efficiency rate of 100% in mouse oocytes. The Polscope can also be used to examine the spindle dynamics, detect spindle morphology, predict chromosome misalignment, and perform spindle transfer.

Animals↗
PubMed1996

Invasive pneumococcal infections in children infected with HIV are not associated with splenic dysfunction.

OBJECTIVES: Children infected with HIV-1 are more likely to acquire infections associated with the encapsulated bacterial pathogens of childhood than their non-HIV-infected peers. The goal of the current study was to determine what proportion of community-acquired, invasive pneumococcal disease in HIV-infected children could be attributed to splenic dysfunction, as measured by enumerating the number of pocked red blood cells (RBCs) in peripheral blood. METHODS: Splenic reticuloendothelial function was assessed semiquantitatively by examining the morphology of the RBCs of 84 children born to HIV-infected mothers using phase interference microscopy. Surveillance of medical records, and a review of the Yale-New Haven Hospital Clinical Microbiology computerized database, revealed that all of the bacterial cultures of blood and cerebrospinal fluid from these patients were positive. RESULTS: Of the 84 children assessed, 70 were infected with HIV (median age 66 months) and 14 were uninfected seroreverters (controls). Sixty-one of the 70 HIV-infected children met the CDC criteria for moderate or severe immunodeficiency and/or moderate or severe symptomatic conditions. Seventeen of the 70 HIV-infected children experienced 23 invasive bacterial infections. Streptococcus pneumoniae was responsible for all 23 infections. The median age at the time of first infection among these 17 subjects was 20 months (range, 10-58 months). There were no episodes of invasive bacterial infections in the remaining 53 HIV-infected children nor among the 14 controls. All 84 children studied, including those with invasive pneumococcal disease, had normal proportions (i.e., < 2%) of pocked erythrocytes in peripheral blood. CONCLUSION: Splenic dysfunction, as measured by the pocked RBC count, does not account for the increased occurrence of invasive pneumococcal disease found in children infected with HIV.

AIDS-Related Opportunistic Infections↗
PubMed1990

Interferon treatment reduces endocytosis of virus and facultatively intracellular bacteria in various cell lines.

Previous studies have shown that interferons (IFNs) specifically interact with a number of cells cultured in vitro and reduce the invasiveness of facultatively intracellular bacteria. IFN treatment also reduced the internalization of vesicular stomatitis virus (VSV) in cell cultures. Here we show that the anti-invasive effect of IFN on bacteria is eliminated in an L-cell variant where its effect on the uptake of vesicular stomatitis virus is lost. The data strongly suggest that the anti-invasive effect of IFN is mediated through inhibition of endocytosis.

Animals↗
PubMed2006

IS911 transpososome assembly as analysed by tethered particle motion.

Initiation of transposition requires formation of a synaptic complex between both transposon ends and the transposase (Tpase), the enzyme which catalyses DNA cleavage and strand transfer and which ensures transposon mobility. We have used a single-molecule approach, tethered particle motion (TPM), to observe binding of a Tpase derivative, OrfAB[149], amputated for its C-terminal catalytic domain, to DNA molecules carrying one or two IS911 ends. Binding of OrfAB[149] to a single IS911 end provoked a small shortening of the DNA. This is consistent with a DNA bend introduced by protein binding to a single end. This was confirmed using a classic gel retardation assay with circularly permuted DNA substrates. When two ends were present on the tethered DNA in their natural, inverted, configuration, Tpase not only provoked the short reduction in length but also generated species with greatly reduce effective length consistent with DNA looping between the ends. Once formed, this 'looped' species was very stable. Kinetic analysis in real-time suggested that passage from the bound unlooped to the looped state could involve another species of intermediate length in which both transposon ends are bound. DNA carrying directly repeated ends also gave rise to the looped species but the level of the intermediate species was significantly enhanced. Its accumulation could reflect a less favourable synapse formation from this configuration than for the inverted ends. This is compatible with a model in which Tpase binds separately to and bends each end (the intermediate species) and protein-protein interactions then lead to synapsis (the looped species).

Binding Sites↗
PubMed1987

The occurrence, recognition and developmental fate of pseudo-multipronuclear eggs after in-vitro fertilization of human oocytes.

The presence of three or more presumed pronuclei in 27 of 884 (3%) in-vitro fertilized human eggs suggested the occurrence of polyspermic fertilization. Detailed examination by differential interference contrast microscopy indicated that approximately 40% of presumed multipronuclear eggs were normally fertilized but contained a cytoplasmic vacuole(s) (pseudo-pronucleus) of pronuclear dimension and gross morphology. Closely timed observations of presumed multipronuclear eggs revealed that pseudo-pronuclei (PPN) can co-migrate and become juxtaposed with true pronuclei, thus presenting the impression of an aberrant fertilization. Fluorescent probe and electron microscopic analyses demonstrated that PPN are enclosed by a plasma membrane and contain neither DNA nor nucleoli. PPN can develop either in the mature oocyte or the newly fertilized egg. Pseudo-multipronuclear eggs progress through the preimplantation stages in an apparently normal fashion and, as demonstrated by one birth and two ongoing pregnancies, are developmentally viable. Criteria for the unambiguous designation of pseudo-multipronuclear eggs are based on: the absence of normal morphodynamic changes associated with pronuclear development; and the failure of nucleoli to appear during the perisyngamic stage. The findings strongly suggest that pseudo-multipronuclear human eggs may represent a class of morphological variants of normally fertilized and developmentally competent eggs.

Cell Nucleolus↗
PubMed1997

Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa.

The combination of specific fluorometric staining and flow cytometry provides a rapid and precise means of assessing the functional status of cells. We sought to utilize this approach to quantify two important seminal characteristics, acrosomal integrity and sperm viability, and to compare these with classical microscopic measurements of acrosomal integrity and sperm motility. Samples of thawed, cryopreserved sperm packaged in 0.5-ml French straws were obtained from 12 Holstein bulls. Classical acrosomal assessments and sperm motility estimates were made using differential interference contrast microscopy. Fluorescent acrosomal probes included LysoTracker Green DND-26 (LYSO-G), fluorescein-labeled peanut agglutinin, the biotinylated isocoumarin serine protease inhibitor Bi-Aca-Aca-OMe-IC that was secondarily labeled with fluorescein-avidin, and rabbit antibodies to bovine acrosin that were secondarily labeled with fluoresceinated anti-rabbit immunoglobulin. The fluorescent probes for sperm viability were a combination of SYBR-14 and propidium iodide (PI) or of SYTO-17 and PI. Significant differences were found among methods and among bulls, but not among straws (n = 3). All four fluorescent measures of acrosomal integrity showed highly significant correlations with both classical measurements. These data indicated that the quality of cryopreserved bovine sperm samples could be readily quantified using a variety of organelle-specific fluorescent staining techniques.

Acrosin↗
PubMed1986

The effects of osmotic tissue dehydration and air drying on morphology and energy transfer in two species of porphyra.

Studies were conducted to document the effects on morphology and energy transfer in photosynthesis of severe tissue dehydration induced either by air-drying or by immersing the tissues of two Porphyra species in hyperosmotic solutions. These studies showed that the dehydration-tolerant intertidal alga, Porphyra perforata J.Ag., was almost unaffected by either of these treatments, while the dehydration-sensitive Porphyra nereocystis Anders. was damaged similary by both treatments. Damage to that sensitive species was characterized by ruptured organelles as seen by interference microscopy as well as by increased fluorescence emission at 682 nanometers emanating from allophycocyanin. These results suggest that a disruption of energy transfer between allophycocyanin and chlorophyll a occurs because of the damage to membranes following tissue dehydration, and that the increase in the yield of phycobilin fluorescence is a good indicator of these phenomena. Thus, air-drying and osmotic-dehydration appear to have similar physiological consequences in a dehydration-sensitive alga but almost no effect in a tolerant species.

Journal Article↗
PubMed2002

Phase-contrast X-ray imaging based on interferometry.

Phase-contrast X-ray imaging including tomographic configuration using a crystal X-ray interferometer is reviewed. The imaging principle based on phase retrieval is described, and some demonstrations showing its high sensitivity are presented on the observations of animal and human cancerous tissues.

Animals↗