Occlusion pressures vs. micropipette pressures in the pulmonary circulation

Because of the discrepancies between the arterial and venous occlusion technique and the micropuncture technique in estimating pulmonary capillary pressure gradient, we compared measurements made with the two techniques in the same preparations (isolated left lower lobe of dog lung). In addition, we also obtained direct and reliable measurements of pressures in 0.9-mm arteries and veins using a retrograde catheterization technique, as well as a microvascular pressure made with the double-occlusion technique. The following conclusions were made from dog lobes perfused with autologous blood at normal flow rate of 500-600 ml/min and pressure gradient of 12 mmHg. 1) The double-occlusion technique measures pressure in the capillaries, 2) a small pressure gradient (0.5 mmHg) exists between 30- to 50-micron arteries and veins, 3) a large pressure gradient occurs in arteries and veins greater than 0.9 mm, 4) the arterial and venous occlusion techniques measure pressures in vessels that are less than 900 microns diam but greater than 50 microns, very likely close to 100 microns, 5) serotonin constricts arteries (larger and smaller than 0.9 mm) whereas histamine constricts veins (larger and smaller than 0.9 mm). Thus three different techniques (small retrograde catheter, arterial and venous occlusion, and micropuncture) show consistent results, confirming the presence of significant resistance in large arteries and veins with minimal resistance in the microcirculation.     

Trichodina myicola n. sp., a Peritrichous Ciliate from the Marine Bivalve Mya arenaria L.

A new ciliate from the marine clam Mya arenaria is described and figured. The form is designated Trichodina myicola with specific characters as follows: Bell-shaped to discoidal; length 31–86 microns; diam. 62–103 microns; diam. denticulate ring 29–46 microns; 26–36 denticles; diam. of basal disk 42–79 microns; adoral ciliary spiral performs slightly more than one turn; posterior ciliature in three closely applied, concentric tiers: an anterior circlet of marginal cilia, a middle circlet of overlapping membranelles, and a posterior circlet of inner cilia; macronucleus C-shaped; micronucleus small, round, lying near outer curvature of left arm of macronucleus; large contractile vacuole present near end of left arm of macronucleus.
Taxonomy in the genus Trichodina is discussed and outlined. Special attention is given to details of the oral ciliature. T. myicola is compared with other members of the genus. A checklist of Trichodina species is presented.     

Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner- or outer-arm dynein: velocity measurements on new types of mutants by an improved method.

To help understand the functional properties of inner and outer dynein arms in axonemal motility, sliding velocities of outer doublets were measured in disintegrating axonemes of Chlamydomonas mutants lacking either of the arms. Measurements under improved solution conditions yielded significantly higher sliding velocities than those observed in a previous study [Okagaki and Kamiya, 1986, J. Cell Biol. 103:1895-1902]. As in the previous study, it was found that the velocities in axonemes of wild type (wt) and a mutant (oda1) missing the outer arm differ greatly: 18.5 +/- 4.1 microns/sec for wt and 4.4 +/- 2.3 microns/sec for oda1 at 0.5 mM Mg-ATP. In contrast, axonemes of two types of mutants (ida2 and ida4) that lacked different sets of two inner-arm heavy chains displayed velocities almost identical with the wild-type velocity. Moreover, axonemes of a non-motile double mutant ida2 X ida4 underwent sliding disintegration at a similar high velocity, although less frequently than in axonemes of single mutants. These observations support the hypothesis that the inner and outer dynein arms in disintegrating axonemes drive microtubules at different speeds and it is the faster outer arm that determines the overall speed when both arms are present. The inner arm may be important for the initiation of sliding. The axoneme thus appears to be equipped with two (or more) types of motors with different intrinsic speeds.     

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.     

海南岛东寨港红树林群落甲烷通量研究

 采用静态箱法对海南东寨港4个站位的5个红树林群落的土壤甲烷通量进行了研究,结果表明林地土壤平均甲烷通量为0.81mg·m-2·d-1。利用聚乙烯袋密闭法测定了6种红树植物叶片的甲烷通量,发现红树植物叶片具有吸收大气甲烷的效应。通过海莲(Bruguiera sexangula)红树林的研究还表明,林地土壤甲烷通量的日变化与林内潮水淹浸状况有关。海莲林不同滩面土壤甲烷通量的差异与土壤含水量有关。土壤甲烷通量的季节差异因植被类型或土壤性质不同而表现为两种形式。     

A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes.

We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth.     

GABA,a new player in the plant mating game

Pollen tubes are guided through female tissues to deliver sperms to the embryo sac. A recent study reveals a GABA gradient along the pollen tube path, which, together with guidance defects in GABA-overaccumulating mutants, implies a role for GABA in regulating pollen tube growth and guidance.     

Motion of cells sedimenting on a solid surface in a laminar shear flow.

Cell adhesion often occurs under dynamic conditions, as in flowing blood. A quantitative understanding of this process requires accurate knowledge of the topographical relationships between the cell membrane and potentially adhesive surfaces. This report describes an experimental study made on both the translational and rotational velocities of leukocytes sedimenting of a flat surface under laminar shear flow. The main conclusions are as follows: (a) Cells move close to the wall with constant velocity for several tens of seconds. (b) The numerical values of translational and rotational velocities are inconsistent with Goldman's model of a neutrally buoyant sphere in a laminar shear flow, unless a drag force corresponding to contact friction between cells and the chamber floor is added. The phenomenological friction coefficient was 7.4 millinewton.s/m. (c) Using a modified Goldman's theory, the width of the gap separating cells (6 microns radius) from the chamber floor was estimated at 1.4 micron. (d) It is shown that a high value of the cell-to-substrate gap may be accounted for by the presence of cell surface protrusions of a few micrometer length, in accordance with electron microscope observations performed on the same cell population. (e) In association with previously reported data (Tissot, O., C. Foa, C. Capo, H. Brailly, M. Delaage, and P. Bongrand. 1991. Biocolloids and Biosurfaces. In press), these results are consistent with the possibility that cell-substrate attachment be initiated by the formation of a single molecular bond, which might be considered as the rate limiting step.     

Steroids and hematopoiesis II. The effect of steroids on in vitro erythroid colony growth: Evidence for different target cells for different classes of steroids

Androgenic steroids and their non-androgenic 5p-H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unitlml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Reincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.     

Aphanomyces infection in juvenile soft-shelled turtle,Pelodiscus sinensis,imported from Singapore

Aphanomyces sp. was isolated from the carapaces of two juvenile soft-shelled turtles with fungal infections imported from Singapore. Their sizes were 2.9–3.5 cm in carapace length. Lesions with integumental necrosis and ulceration looked like white cotton. The fungus exhibited slow growth, hyphae were 7.5–15 μm in diam, coarse, and abundantly branched. Zoosporangia observed in the isolate were complex, its entire thallus being converted into zoosporangial units, with short or long lateral evacuation tubes, and isodiametric, 100–500 μm in length. Clusters of zoospores were also produced at the terminals of hyphae. The production of the primary zoospores was achlyoid. The primary encysted zoospores were spherical, 10–15 μm in diam. No sexual stages were observed on a hemp seed incubated in sterile tap water. The optimal temperature for the fungus was 30° C.     

Maximum rates of cooling by three programmable freezers, and the potential relevance to sperm cryopreservation

Maximum rates of cooling for the Asymptote EF 100 and the Cryologic CL8800 temperature controller with either a standard or fast chamber were determined and viewed in the context of sperm cryopreservation. All three systems use liquid nitrogen to cool the plate or chamber which would hold the sample, opposed by a variable amount of heat from an internal heater. Maximum rates of cooling for all systems were a function of the temperature gradient between the liquid nitrogen and the plate/chamber and at a plate/chamber temperature of 15 degrees C were 16.5 degrees C/min, 13.3 degrees C/min and 8.0 degrees C/min for the Asymptote EF100, Cryologic fast and slow chambers respectively. These machines are not suited to the freezing of sperm from species requiring rapid rates of cooling, an important consideration when planning to purchase a piece of equipment for this application, and scientists are advised to discuss specifically their requirements with prospective suppliers.     

Specific adhesion of glycophorin liposomes to a lectin surface in shear flow.

The adhesion of cells to other cells or to surfaces by receptor-ligand binding in a shear field is an important aspect of many different biological processes and various cell separation techniques. The purpose of this study was to observe the adhesion of model cells with receptor molecules embedded in their surfaces to a ligand-coated surface under well-defined flow conditions in a parallel plate flow chamber. Liposomes containing glycophorin were used as the model cells to permit a variation in the adhesion parameters and then to observe the effect on adhesion. A mathematical model for cell sedimentation was created to predict the deposition time and the velocity preceding adhesion for the selection of experimental operating conditions and the methods useful for data analysis. The likelihood of cell attachment was represented by a quantity called the sticking probability which was defined as the inverse of the number of times a liposome made contact with the surface before attachment occurred. The sticking probability decreased as the cell receptor concentration was lowered from approximately 10(4) to 10(2) receptors per 4-microns diam liposome and as the shear rate increased from 5 to 22 s-1. The effect of the wall shear rate and particle diameter on detachment of liposomes from a surface was also observed.     

Locomotion of human bone marrow and peripheral blood leukocytes is associated with maturational stage and altered in malignancy.

Using high-resolution phase contrast time-lapse microcinematography, slow movements (0.8-2.0 microns/minute) of human myeloblasts, monoblasts, and megakaryocytes can be recorded. Upon maturation to promyelocytes, motility is lost until cells have reached the stage of metamyelocytes (0.4 microns/minute). Motility increases sharply following maturation into segmented neutrophils (20.4 microns/minute). Monocytes and promonocytes display a mean track velocity of 7.1 microns/minute. The distribution of lymphocyte velocities is not bell-shaped but shows three maxima of 2.1 microns/minute, 7.8 microns/minute, and 18.4 microns/minute. Atypical lymphocytes from patients with infectious mononucleosis belong to the fast group, whereas lymphocytes activated in vitro by mitogens belong to the slow group. Red blood cell precursors from normal human bone marrow do not move actively. In contrast, erythroleukemic blasts show a motility comparable to normal myeloblasts. Similarly, acute promyelocytic leukemia cells move at 6.7 microns/minute, while their normal counterparts are sessile. Increased motility is also observed in blast cells from a variety of acute myelogenous and lymphoblastic leukemias.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. III. Plasmalemma, "membranosomas".

Ultrastructure of the plasma membranes of cytolytical T lymphocytes (CTL) in their interaction with target cells (TC) was studied. Thirty to sixty minutes after the beginning of interaction shedding of the CTL plasma membrane was observed: its fragments shedded from a local (50–100 nm in diam.) area on the lymphocyte surface at the site opposite to the CTL contact region with TC. Oval structures of high electron density 10 to 40 nm in diam. were detected on the CTL surface. We designated them as “membranosomas” (MS). MS were in close apposition to the inner surface of the plasma membrane and showed projections of 2 to 3 nm in diam. and 5 to 6 nm long towards the outer surface of the plasma membrane. MS were separated from the CTL surface during clasmatosis or as component parts of “shedding” plasma membranes.     

Lamprothamnium, a Euryhaline Charophyte: III. IONIC FLUXES AND COMPARTMENTAL ANALYSIS AT STEADY STATE

Ionic fluxes, membrane potentials and permeabilities of theplasmalemma and tonoplast to K¹, Na¹ and Cl^– have beenexamined under steady-state conditions in the brackish-watercharophyte. Lamprothamnium papulosum. Cells were placed in oneof three aerated solutions of artificial seawater (500, 750,1050 mosmol kg^–1). Mean vacuolar potentials were –175,–166 and –157 mV respectively in the three solutions.Compartmental analysis of fluxes indicated that sodium and potassiumwere moved from the cytoplasm to both the vacuole and the externalsolution against the electrochemical gradient, whereas inwardmovements of chloride from the external solution and the cytoplasmwere against the gradient. The Na/K ratio in the cytoplasm wasgreater than one. The low passive permeability of the tonoplastresulted in only a slow loss of ions, particularly K¹ from thevacuole. These results are discussed in relation to osmoticregulation under steady-state conditions. Key words: Lamprothamnium, onic flux, ompartmental analysis     

Hydrotropism in abscisic acid,wavy, and gravitropic mutants of Arabidopsis thaliana

We have developed experimental systems to study hydrotropism in seedling roots of Arabidopsis thaliana (L.) Heynh. Arabidopsis roots showed a strong curvature in response to a moisture gradient, established by applying 1% agar and a saturated solution of KCl or K(2)CO(3) in a closed chamber. In this system, the hydrotropic response overcame the gravitropic response. Hydrotropic curvature commenced within 30 min and reached 80-100 degrees within 24 h of hydrostimulation. When 1% agar and agar containing 1 MPa sorbitol were placed side-by-side in humid air, a water potential gradient formed at the border between the two media. Although the gradient changed with time, it still elicited a hydrotropic response in Arabidopsis roots. The roots curved away from 0.5-1.5 MPa of sorbitol agar. Various Arabidopsis mutants were tested for their hydrotropic response. Roots of aba1-1 and abi2-1 mutants were less sensitive to hydrotropic stimulation. Addition of abscisic acid restored the normal hydrotropic response in aba1-1 roots. In comparison, mutants that exhibit a reduced response to gravity and auxin, axr1-3 and axr2-1, showed a hydrotropic response greater than that of the wild type. Wavy mutants, wav2-1 and wav3-1, showed increased sensitivity to the induction of hydrotropism by the moisture gradient. These results suggest that auxin plays divergent roles in hydrotropism and gravitropism, and that abscisic acid plays a positive role in hydrotropism. Furthermore, hydrotropism and the wavy response may share part of a common molecular pathway controlling the directional growth of roots.     

Gravitational and magnetic activation methods applied to cultured cells (LK 35.2)

Monoclonal antibody 10.2-16 is directed toward the mouse class II major histocompatibility complex gene product 1-Ak expressed on the cell line LK35.2. Instead of activating cells by fluorophor we used (acrylamide-coated) heavy and magnetic microspheres of 0.6 micron in radius. These microspheres are chemically coupled (carbodiimide method) with the antibody toward the surface antigen. The cells are observed through a microscope with horizontal alignment, as they sediment in a (temperature controlled) tube with square cross-section. Stokes Law allows the determination of the density of cells (first alone) using viscosity and density of Dulbecco's modified Eagle's Medium together with the observed mean sedimentation velocity (66 microns/min) and a mean diameter of 10 microns. We found a density of 1.0558 +/- 0.0028 g/cm3 at 10 degrees C. Independently, thinly coated, heavy (and magnetizable) microspheres with the cited antibody are attached to cells and observed likewise. The increased sedimentation velocity permits us to show that the cells were fully covered with microspheres (290 per cell). A magnetic field gradient opposing gravity moved these cells against gravity with two different mean velocities, 340 microns/min and 850 microns/min. The higher velocity resulted in 290 particles per cell, the lower one in 130 particles per cell. The limits for the expansion of this method to smaller particle sizes (down to 10 nm) are evaluated.     

Density gradient system. I. Formation and fractionation of density gradients

The instrumentation of a precise yet flexible system for the formation and fractionation of linear density gradients is described. The use of a special peristaltic pump and gradient-forming chambers permits the rapid formation of both dilute and concentrated solutions into ca. 5 to 300 ml of gradient. This gradient can be delivered to a single centrifuge tube or equally apportioned to as many as 24 tubes.The special peristaltic pump, which is described in the paper that follows, delivers the concentrated solution into the gradient-forming chamber as well as conveying the gradient from the chamber to the special centrifuge tube. A capillary in the wall of the tube conveys the gradient to the bottom, where it is dispersed into 18 equidistant orifices. This dispersion permits the rapid formation and fractionation of the gradient without disruption. A presettable counter coupled to the peristaltic pump permits the gradients to be apportioned into equal volume fractions.Results obtained with both dilute and concentrated gradients are presented.     

Observations on the response of human spermatozoa to gravity, boundaries and fluid shear

Human sperm motility response to three mechanical stimuli, gravity, fluid flow shear and rigid boundaries, was measured in a tube of 310 X 400 microns calibre. Data were gathered by cine recordings at various focussing levels d across the tube and analysed with a computerized image analysis system. The most influential stimulus was the tube wall near (more than 'at') which the swimmers tended to accumulate, leaving the fluid beyond 100 microns from the wall (d = 100) vacant of motile spermatozoa. The boundary effect was evident as soon as the spermatozoa could be viewed after loading, and accumulation, measured as frequency, as a function of d did not change with time t. This response was not significantly altered by the addition of laminar flow with a centre line velocity of about 400 microns/sec. In flow shear, spermatozoa aligned positively (in the flow direction) at the wall but negatively by about 30 microns from the wall where the velocity gradient (= shear rate) was about 3.5 sec-1. The response to gravity was relatively weak with 11 spermatozoa positive (swimming downwards) for each 9 negative. Neither the boundary effect nor the 'rheotaxic' effect were influenced by gravity as there was no statistical difference in orientation or distribution patterns between vertically and horizontally flowing suspensions. It is suggested that the boundary effect cannot be ignored in in-vitro manipulations, particularly when spermatozoa are observed or extracted. Its importance in vivo lies in the degree to which the tubes transporting motile spermatozoa seem to have mechanisms for reversing the wall accumulation tendency.     

Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.