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PubMed1990

A method for isolation and fluorescent labeling of rat neutrophils for intravital microvascular studies.

A method for the isolation, fluorescent labeling, and reinfusion of rat neutrophils for in vivo investigation of white blood cell function in the microcirculation is described. The cell surface morphology and function of the labeled and unlabeled neutrophils were compared in vitro and in vivo. The morphology of the labeled and unlabeled cells was visually assessed by differential interference contrast microscopy before and after exposure to the chemotactic peptide FMLP. Labeled and unlabeled cells were morphologically similar under normal and stimulated conditions. The chemotactic responsiveness of the labeled and unlabeled cells was evaluated in vitro by quantifying the movement of cells across a porous membrane in response to FMLP. No significant difference in chemotactic responsiveness was found. The in vivo behavior of the labeled neutrophils was quantitatively studied by examining their interactions with the venular endothelium in the rat cremaster muscle. Rolling velocity for labeled neutrophils and unlabeled white blood cells was measured at different flow velocities and wall shear rates. No difference was found between the labeled and unlabeled cells. It is concluded that this isolation and labeling procedure yields cells possessing normal morphology and function. These labeled neutrophils may be useful for in vivo study of their behavior in the microcirculation.

Animals↗
PubMed1988

In vitro excystation and subsequent axenic growth of Giardia lamblia.

Six fresh Giardia lamblia strains were isolated by in vitro excystation and subsequent culturing of excysted parasites in bile-supplemented BI-S-33 medium. The cysts passed in faeces appeared to differ in structure when observed using differential interference contrast microscopy. Sometimes the enclosed organisms were closely applied to the cyst wall; in most stool specimens, however, the parasites were separated from the cyst wall by a space. Cysts with parasites closely applied to the wall were the most viable type, with excystation rates up to 90%. Cysts with organisms detached from the wall displayed low excystation rates, 0-15%. During exposure to the induction solution of hydrochloric acid (pH 2), parasites initially closely applied to the cyst walls completely detached, and after transfer into culture medium vigorous flexing movements of the organisms were observed. Stool samples from 42 symptomatic and asymptomatic giardiasis patients were examined; in 26 of the samples parasites hatched, and 6 new strains were established in axenic culture.

Animals↗
PubMed1978

Layered membranes: a diffusion barrier to gases in teleostean swimbladders.

Flattened cells are dispersed in an overlapping fashion within the submucosa of closed (physoclistous) swimbladders. Within the cells are located round platelets best seen by phase or interference type microscopy. At the electron microscope level the platelets are revealed as repeated, numerous layers of membranous discs. Each disc is a bilayer with an enlarged rim. The bilayer is strongly and preferentially reactive to osmium tetroxide. Swimbladder walls are known to be highly resistant to passage of gases. The randomly disposed platelets with the highly ordered layering of membranes may provide that physical resistance to gas movement.

Air Sacs↗
PubMed1983

Newt lungs: a versatile system for the study of mucociliary transport.

Lungs of the newt, Taricha granulosa, provide a versatile system for studying mucociliary transport. The mucociliary epithelium is restricted to a ventral strip of epithelium which extends the entire length of the lung. The transport process can be studied in the isolated lung or in a variety of functional components derived from it. These components include: (1) isolated sheets of the mucociliary epithelium, (2) flattened epithelial sheets grown in long term primary culture, (3) dissociated ciliated and mucous cells, (4) functional, demembranated ciliary tufts, and (5) isolated populations of discrete, demembranated axonemes. The latter two models can be reactivated with MgATP and made to beat at frequencies comparable to those measured in intact cells. Featuring large cells (25-30 microns in diameter) and cilia (12-13 microns in length) in comparison to other mucus-transporting systems, coupled with the ability to perform 'physiological' experiments at ambient temperatures, newt lungs afford a convenient system to study mechanisms involved in the control of ciliary beat frequency, waveform and coordination at several levels of organization.

Animals↗
PubMed1992

Sertoli cells isolated from adult 2,5-hexanedione-exposed rats exhibit atypical morphology and actin distribution.

Sertoli cells were isolated from 2,5-hexanedione (2,5-HD)-exposed, cryptorchid and 21-day-old rats in order to examine alterations in in vitro Sertoli cell transferrin secretion, germ cell adhesion, in vitro morphology, and cytoskeletal organization which might be involved in the irreversibility of 2,5-HD-induced testicular injury. Sertoli cells isolated from 21-day-old, cryptorchid and 2,5-HD-exposed rats exhibited similar transferrin secretion as measured using an enzyme-linked immunosorbent assay. Germ-cell adhesion was measured using [3H]leucine-labeled immature rat germ cells and revealed similar levels of germ-cell binding in Sertoli cell cultures isolated from the three groups of rats. Differential interference contrast microscopy demonstrated that Sertoli cells isolated from 2,5-HD-exposed rats possessed an atypical spindle shape and long cytoplasmic processes. The immunofluorescent distribution of tubulin and vimentin corresponded with the morphological appearance of the cells with well-defined microtubule and intermediate filament networks which, in the cells isolated from 2,5-HD-exposed rats, extended into the cytoplasmic processes. Rhodamine-conjugated phalloidin-labeled actin stress fibers were decreased in density within the 2,5-HD-exposed rat Sertoli cells. The altered morphology and distribution of actin filaments within Sertoli cells isolated from adult 2,5-HD-exposed rats may reflect an underlying insult which is involved in the irreversible nature of 2,5-HD intoxication.

Actins↗
PubMed1991

Requirements for nuclear localization and supramolecular assembly of a baculovirus polyhedrin protein.

This study defines the requirements for the nuclear localization, stable nuclear association, and supramolecular assembly of a baculovirus polyhedrin protein in lepidopteran insect cells. Fragments of the polyhedrin protein were genetically fused to two different nonnuclear reporter proteins and the intracellular distribution of the fusion proteins was analyzed in infected insect cells. Analysis by indirect immunofluorescence showed that the domain between amino acids 30 and 57 could mediate nuclear localization of polyhedrin. However, biochemical fractionation experiments showed that this domain was not sufficient for a detergent-stable association of polyhedrin with the nucleus. This required a slightly larger domain, between amino acids 30 and 110. Differential interference-contrast microscopy showed that the supramolecular assembly of polyhedrin into nuclear occlusion-like particles required the domain between amino acids 19 and 110. The most likely candidate for a minimal nuclear localization signal was the sequence KRKK, located between amino acids 32 and 35. Therefore, oligonucleotide-directed mutagenesis was used to change this sequence to NGNN and the intracellular distribution of the mutant protein was analyzed. The results showed that the mutant protein was predominantly localized in the cytoplasm of infected cells, where it assembled into large, cubic, occlusion-like particles. Thus, the KRKK sequence is necessary for the nuclear localization of polyhedrin, but nuclear localization is not required for its supramolecular assembly into occlusion-like particles.

Amino Acid Sequence↗
PubMed1987

A whole mount method for sequential analysis of photoreceptor and ganglion cell topography in a single retina.

Photoreceptors (PR) in human and monkey retina are visible in whole mounts cleared with glycerol or dimethyl sulfoxide and viewed with Nomarski differential interference contrast microscopy. These preparations substantially decrease the large tissue volume changes associated with dehydration and sectioning and reveal many details of PR organization and cytology with great clarity. Tissue may be subsequently stained to reveal ganglion cells so that the topography of both cell types may be studied in the same retina.

Animals↗
PubMed1984

Optical shape change analysis in stirred and unstirred human platelet suspensions. A comparison of aggregometric and stopped-flow turbidimetric measurements.

To monitor the discoidity of platelets in an aggregometer, a relative, stirring-dependent change in extinction (delta ES) is defined. delta ES is large in highly discoid and small in ADP-activated platelets. The platelet shape change, as monitored by the changes in delta ES, not only precedes ADP-induced aggregation but also outlasts disaggregation. In unstirred samples, ADP induces insignificant changes in extinction in the aggregometer but biphasic changes in extinction in a "zero degree" stopped-flow turbidimeter. This discrepancy apparently arises from differences in the amount of scattered light collected by the optical systems and from wavelength-dependent differences in sensitivity. Similar progress curves were observed in both instruments for the biphasic changes in extinction which accompany the release reaction induced by thrombin or concanavalin A in pre-sphered platelets. The aggregometer is advantageous for monitoring rheooptical effects of the asymmetric platelets while the stopped-flow laser turbidimeter is superior in quantifying the changes in extinction according to the light scattering theory.

Adenosine Diphosphate↗
PubMed1994

Nitric oxide donors inhibit platelet spreading on surfaces coated with fibrinogen but not with fibronectin.

We have studied the effect of nitric oxide (NO) on the interaction of washed human platelets with fibrinogen or fibronectin adsorbed on glass. Platelet contacts were visualized by interference reflection microscopy at 37 degrees C in Tyrode's solution. The areas of spread platelets were measured, using digital video image processing techniques, at times up to 30 minutes after initial surface platelet contact. On fibrinogen spreading was inhibited by NO donors in the order of potency: S-nitroso-acetylpenicillamine > sodium nitroprusside > S-nitroso-glutathione. The inhibitory action of NO donors was prevented by Oxy-haemoglobin, confirming that this inhibition was due to the release of NO. In contrast, NO donors had virtually no effect on platelet spreading on fibronectin. Platelet adhesion to fibrinogen is mediated by GP IIb IIIa and the vitronectin receptor (VNR), while that to fibronectin is via GP IIb IIIa, VNR, Ic IIa and VLA-6. Our results thus indicate that NO inhibits adhesion mediated by GP IIb IIIa and/or VNR but not by the two other receptors. The mechanism of this receptor-specific inhibition of platelet adhesion remains to be elucidated.

Adsorption↗
PubMed1985

Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon.

Cytoplasmic filaments, separated from the axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, support the directed movement of organelles in the presence of ATP. All organelles, regardless of size, move continuously along isolated transport filaments at 2.2 +/- 0.2 micron/sec. In the intact axoplasm, however, movements of the larger organelles are slow and saltatory. These movements may reflect a resistance to movement imposed by the intact axoplasm. The uniform rate of all organelles along isolated transport filaments suggests that a single type of molecular motor powers fast axonal transport. Organelles can attach to and move along more than one filament at a time, suggesting that organelles have multiple binding sites for this motor.

Adenosine Triphosphate↗
PubMed1985

Single microtubules from squid axoplasm support bidirectional movement of organelles.

Single filaments, dissociated from the extruded axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, transport organelles bidirectionally. Organelles moving in the same or opposite directions along the same filament can pass each other without colliding, indicating that each transport filament has several tracks for organelle movement. In order to characterize transport filaments, organelle movements were first examined by video microscopy, and then the same filaments were examined by electron microscopy after rapid-freezing, freeze-drying, and rotary-shadowing. Transport filaments that supported bidirectional movement of organelles are 22 nm to 27 nm in diameter and have a substructure indicative of a single microtubule. Immunofluorescence showed that virtually all transport filaments contain tubulin. These results show that single microtubules can serve as a substratum for organelle movement, and suggest that an interaction between organelles and microtubules is the basis of fast axonal transport.

Animals↗
PubMed1990

Concentration of membrane antigens by forward transport and trapping in neuronal growth cones.

Formation of the nervous system requires that neuronal growth cones follow specific paths and then stop at recognition signals, sensed at the growth cone's leading edge. We used antibody-coated gold particles viewed by video-enhanced differential interference contrast microscopy to observe the distribution and movement of two cell surface molecules, N-CAM and the 2A1 antigen, on growth cones of cultured cortical neurons. Gold particles are occasionally transported forward at 1-2 microns/s to the leading edge where they are trapped but continue to move. Concentration at the edge persists after cytochalasin D treatment or ATP depletion, but active movements to and along edges cease. We also observed a novel outward movement of small cytoplasmic aggregates at 1.8 microns/s in filopodia. We suggest that active forward transport and trapping involve reversible attachment of antigens to and transport along cytoskeletal elements localized to edges of growth cones.

Actins↗
PubMed1993

A comparison of 1- and 2-cell ova production by F2 50% Meishan versus F1 White line gilts.

The objective of this study was to compare recovery of pronuclear and 2-cell ova from F2 50% Meishan (MX) gilts versus F1 White line (L42) gilts. Sexually mature MX and L42 gilts were allocated across 2 treatments: Super (MX:n=9; L42:n=10) and Control (MX:n=6; L42:n=5) in a 2 x 2 factorial experiment. Allyl trenbolone (AT) was used to synchronize estrus in all gilts. Super gilts were given pregnant mare serum gonadotropin (PMSG: 1250 IU) at 24 h after AT withdrawal. Eighty-five hours after PMSG administration, all Super gilts received 750 IU of human chorionic gonadotropin (hCG). Super gilts which exhibited estrus within 24 h of hCG administration (MX-Super: n=6; L42-Super: n=5) and all Control gilts were bred naturally to Line 3 boars at 12 and 24 hours after the onset of estrus. Ova were recovered from Super gilts between 60 and 64 h after hCG and Control gilts at 48 h after the onset of estrus. All 1- and 2-cell ova were centrifuged at 15000 x g and observed using differential interference contrast microscopy. The mean ovulation rate was greater (P<0.05) for both MX-Super and L42-Super gilts in comparison to their respective Control groups. No differences were detected in the mean ovulation rate (P>0.38) or the mean number of 1- and 2-cell ova recovered (P>0.50) between MX-Super and L42-Super gilts. The proportion of 1- and 2-cell ova which exhibited visible pronuclei or nuclei was also similar among MX-SUPER and L42-SUPER gilts. This study demonstrates that MX gilts respond/perform comparably to L42 gilts with respect to estrus synchronization, superovulation, ova yield, and the ease of visibility of pronuclei or nuclei in the ova.

Journal Article↗