Lipolysis of serum-activated triacylglycerol at the surface of J774.1 macrophages

Summary Cultured mouse (J774.1) macrophages accumulated triacylglycerol, but no cholesteryl ester or cholesterol, when incubated in albumin-poor medium with serum-activated lipid particles containing 84 mol% trioleoylglycerol and 9 mol% cholesteryl oleate. Accumulation of triacylglycerol by cells was associated with hydrolysis of particulate triacylglycerol to fatty acid and glycerol. Both acyl and glyceryl moieties of particulate triacylglycerol were recovered in cellular triacylglycerol with a molar ratio of 3.6. The cells also accumulated fatty acid and monoacylglycerol. Whether acylglycerol was taken up as a single molecular species, such as monoacylglycerol, or as several species can not be determined by the present findings. Macrophages incubated with lipid particles for 24 h had many lipid particles attached to cell surfaces and numerous intracellular lipid droplets. The surface film of attached particles was continuous with the outer leaflet of plasma membrane of the cells. Particles partially depleted of core triacylglycerol and collapsed surface films were found attached to surfaces of macrophages. There was no morphological evidence that lipid particles were taken up intact by cells, through endocytosis or phagocytosis. Macrophages incubated with lipid particles also contained intracellular lamellar structures. They varied in size and shape, and were located in the periphery of cells, sometimes near lipid droplets and endoplasmic reticulum. Only 3% of the lamellar structures were associated with lysosomes, indicating they probably were not of lysosomal origin. Lipid particles attached to cells decreased in size and number, and lamellar structures developed at the surface of particles, or replaced the particles, when glutaraldehyde-fixed specimens were incubated at 25° C, demonstrating lipolytic activity at the surface of macrophages. Our findings suggest that particulate triacylglycerol was hydrolyzed by lipoprotein lipase at the surface of macrophages, and that fatty acid and monoacylglycerol formed by lipolysis were transported directly into the cells to be reesterified. When lipolytic products were taken up faster than they could be utilized, they accumulated as lamellar structures in the cells.Abbreviations MEM Eagle's alpha modification of minimum essential medium     

Partial and complete detachment of neutrophils and eosinophils from schistosomula: evidence for the establishment of continuity between a fused and normal parasite membrane

Neutrophils and eosinophils adhering to the surface of schistosomula of Schistosoma mansoni have been partially or completely detached with hypertonic sucrose or by pipetting. The sucrose-treated neutrophils are attached only in areas where there are pentalaminar fusions between the neutrophil and tegumental membranes, suggesting that these fusions attach the cells to the parasites. Pipetting breaks many of the attached cells. In thin section, the tegumental membrane underlying these cells is seen to be pentalaminar. By freeze-fracture techniques, modified attachment areas are found. The edge zone often appears as a single strand of intramembrane particles (IMPs) on the P2 face and as a groove on the E2 face. The edge zone may also have large discontinuities, in which case it no longer separates membrane faces of unequal IMP density from one another. In addition, the IMPs on the IMP- rich areas become aggregated and surrounded by craters in the membrane. These experiments suggest that the fusions may be the mechanism by which the parasite acquires some host membrane components on its surface. On the other hand, eosinophil plasma membranes are seen adhering to a layer of electron-dense material on the parasite after the cells have been disrupted by pipetting. This suggests that eosinophils adhere to the parasite surface through their discharged granule material and not by membrane fusions.     

OBSERVATIONS ON THE MODE OF RELEASE OF HERPES VIRUS FROM INFECTED HELA CELLS

The development of a well adapted strain of herpes virus has been studied in HeLa cells using thin sectioning techniques for electron microscopy. Particular attention was directed to events in the cytoplasm and certain new features were observed. Profuse immature particles with a nucleoid and single limiting membrane were present in the nuclei of infected cells, often in crystalline array; morphologically indistinguishable immature particles were also found very frequently in the cytoplasm. Cells with such particles were intact and well preserved, and contained smooth vacuoles apparently derived from the Golgi component of the endoplasmic reticulum. The cytoplasmic particles escaped from the cells by bulging out as buds through the cell membrane or through that of the cytoplasmic vacuoles until they were attached only by a pedicle and then became free. During this process the particles were gradually enclosed by the membrane through which they passed and carried a coat of it with them as they matured. After permanganate fixation the triple-layered structure of the cell membrane and vacuolar membranes was evident and was identical with that of the outer coat of the mature virus. These findings are discussed both in relation to different types of virus structure and to function in the endoplasmic reticulum and cell membrane.     

Cell sedimentation with gravity activation

Murine monoclonal antibody T101 has been coupled to thinly polymer-coated heavy alloy particles (LaMn2Ge2). These conjugates are coupled to cultured cells of the human T-cell leukemia line RPMI 8402 (T8402). The sedimentation velocities of cells, of particles, and of cells with particles attached are measured. After determining the mean radii of cells, of particles, and of cells with particles attached, one may compute a mean number of 33 particles attached to a cell. Independently one may compute a mean number of 144 particles/cell for surface saturation. The Appendix handles the underlying theory in three parts: number of particles/cell, saturation number of particles/cell, and resolution for gravity activation. Regarding the latter, cell radii from 4 to 10 microns and particle radii from 0.01 to 1 micron are considered.     

The function of tail fibers in triggering baseplate expansion of bacteriophage T4

Normal particles of bacteriophage T4 have six long tail fibers attached to a hexagonal baseplate. T4 particles having various complements of tail fibers were prepared by in vitro addition of fibers to fiberless particles, and the infectivity of the particles was determined. Particles having fewer than six fibers (partially fibered) were found to have a decreased probability of infection. Partially fibered particles having T4 fibers were completed by addition of T6 fibers, and the infectivity was determined on a host that lacked the T6 tail fiber receptor. Attachment of the additional fibers increased the infectivity even though the T6 fibers could not bind to the host cell. The infectivity of particles having mixtures of T4 and T6 fibers was determined on cells having only one type of receptor. The results indicated that particles bound by only three fibers have a low probability of infection. The effect of thermolabile baseplate mutations was also examined. Studies of partially fibered particles and particles with mixtures of fibers indicated that particles with altered baseplates have a less stringent requirement for binding of the tail fibers for infection.     

Morphology of defense glands of the opilionids (daddy longlegs) Leiobunum vittatum and L. flavum (Arachnida: Opiliones: Palpatores: Phalangiidae)

Opilionid defense glands consist of 0.5 × 0.9-mm sacs attached to the underside of low tubercles located on the dorsal side of the cephalothorax, posterior to the first pair of legs. Each gland opens via an elongated slit, located in the posterior floor of a crater that is situated at the summit of the tubercle. The center of the sac, called the reservoir, is lined by a cuticle consisting of epicuticle and endocuticle which is continuous through the slit with the exoskeleton. The layers of cuticle vary in thickness with different locations in the gland. A hemocoelomic (basement) membrane, 0.5–1, μ thick, forms the boundary between glandular cells and hemocoel. The gland has a nonsecretory portion consisting only of cuticle-supporting cells and a secretory portion consisting of secretory and cuticle-supporting cells. The cuticle lining the reservoir in the secretory area is broached by many cuticle-lined ductules, each of which drains an isolated intercellular space called the intercalated cistern. This in turn drains microvilli-lined canaliculi located between and extending into secretory cells. The cisterns are devoid of microvilli. Secretory cell cytoplasm contains a Golgi apparatus, many free ribosomes, rough endoplasmic reticulum (RER), two types of granules (speckled and dense), and mitochondria. Speckled granules are partially filled with fairly large particles and are found in association with the Golgi apparatus. They also surround canaliculi into which they empty. Dense granules are packed with very small particles, have a gray homogeneous appearance, and are scattered throughout the cytoplasm. Mitochondria containing matrix granules tend to scatter throughout the cytoplasm but are concentrated around canaliculi.     

ATTACHMENT OF RIBOSOMES TO MEMBRANES DURING POLYSOME FORMATION IN MOUSE SARCOMA 180 CELLS

Addition of nutrients to starved mouse S-180 cells leads to rapid conversion of ribosomal monomers to polysomes. During this process, a portion of the ribosomes originally found in the 17,000 g (10 min centrifugation) supernatant of cell lysates becomes firmly attached to structures sedimenting at 500 g (5 min centrifugation). Electron microscopy of sections of the intact cells showed the change from randomly distributed ribosomal particles to clusters. Association with membranes also became evident. The material sedimenting at 500 g comprised nuclei enclosed in an extensive endoplasmic reticulum (ER) network. This fraction prepared from recovering cells showed numerous ribosome clusters associated with the ER network. The appearance of many of these clusters indicated that the ribosomal particles were not directly bound to the membranes. RNase treatment released about 40% of the attached ribosomes as monomers, and ethylenediaminetetraacetic acid released 60% as subunits. It is suggested that during polysome formation a portion of the ribosomes becomes attached to the membranes through the intermediary of messenger RNA.     

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection.

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.     

The P2 Protein of Rice Dwarf Phytoreovirus Is Required for Adsorption of the Virus to Cells of the Insect Vector

Intact particles of rice dwarf phytoreovirus adsorbed to and entered monolayer-cultured cells of the insect vector Nephotettix cincticeps and multiplied within the cells. Particles that lacked the P2 protein neither attached to nor infected such cells. Furthermore, P2-free particles obtained from a transmission-competent isolate of the virus were unable to infect insect vectors that had been allowed to feed on these virus particles through a membrane. However, when such virus particles were injected into insects via a glass capillary tube they successfully infected the insects, which became able to transmit the virus. These results support the hypothesis that, while P2-free particles can neither interact with nor infect cells in the intestinal tract of the insect vector, they do retain the ability to infect such cells when physically introduced into the hemolymph by injection.     

Actin filament-membrane attachment: are membrane particles involved?

The association of actin filaments with membranes is an important feature in the motility of nonmuscle cells. We investigated the role of membrane particles in the attachment of actin filaments to membranes in those systems in which the attachment site can be identified. Freeze fractures through the end-on attachment site of the acrosomal filament bundles in Mytilus (mussel) and Limulus (horseshoe crab) sperm and the attachment site of the microvillar filament bundles in the brush border of intestinal epithelial cells were examined. There are no particles on the P face of the membrane at these sites in the sperm systems and generally none at these sites in microvilli. In microvilli, the actin filaments are also attached along their lengths to the membrane by bridges. When the isolated brush border is incubated in high concentrations of Mg++ (15 mM), the actin filaments form paracrystals and, as a result, the bridges are in register (330 A period). Under these conditions, alignment of the particles on the P face of the membrane into circumferential bands also occurs. However, these bands are generally separated by 800-900 A, indicating that all the bridges cannot be directly attached to membrane particles. Thus membrane particles are not directly involved in the attachment of actin filaments to membranes.     

Structure and Role of the Renette Cell and Caudal Glands in the Nematode Sphaerolaimus gracilis (Monhysterida)

Ultrastructure of the renette cell and caudal glands was studied in the free-living aquatic nematode Sphaerolaimus gracilis. The renette cell occurred posterior to the esophageal-intestinal junction and opened through an ampulla to a ventral pore behind the nerve ring. The caudal gland system of the tail consisted of two gland cells opening through separate pores and 2 to 3 other gland cells of a different type opening through a common pore. The renette cell and the two caudal gland cells were similar and both contained secretory granules, 0.5-1.5 μm in diameter. The material released attached the nematode to the substrate. The renette ampulla was surrounded by a specialized cell, the ampulla cell, which had characteristics of myoepithelium. A plug or valve structure connected to the ampulla cell may regulate the output of the secretory material. The ampulla cell is able to contract and thus is probably under direct neuronal control. Other cells in the renette ampulla region of body cavity were termed supporting cells. Living, cold-relaxed nematodes were attached to sediment particles in the renette pore region and at the tail tip. Release from sediment particles was mechanical at the renette cell discharge site but appeared to be chemical at the caudal gland. In behavioral experiments, nematodes in a water current had the ability to release a thread from the caudal glands while maintaining contact with a sediment particle attached to the tail end. If the thread was strong enough, it also could be used to change location. Nematodes anchored by the thread from the caudal glands to a sediment particle could float in water currents until they attached themselves to another sediment particle with the help of secretions from the renette cells.     

Kinetic model of elemental sulfur oxidation by Thiobacillus thiooxidans in batch slurry reactors

The kinetics of sulfur oxidation by T. thiooxidans has been studied in a batch well-mixed reactor and in shaker flasks. A mathematical model is proposed, which considers the attachment of the cells onto the sulfur particles' surface following Freundlich isotherm, growth of the attached bacteria, and growth inhibition by sulfates accumulation. Best-fit values of the model parameters have been calculated from the experimental data. Results show that the addition of dimethyl-dichloro-silane in the aerated reactor to prevent the formation of foam reduces the maximum specific growth rate of attached bacteria, probably because of the resulting changes in surface properties of the sulfur particles. The other model parameters are not significantly affected. The formation of clusters of sulfur particles has been observed at an initial sulfur concentration of 5% . This phenomenon reduces the rate of sulfur conversion due to the reduction of the total surface area of the particles, and the model therefore over-estimates the formation of sulfates. At lower initial sulfur concentration, the phenomenon has not been observed and the model simulations are then satisfactory.     

Nutritional control of copper uptake in the cyanobacteriumNostoc calcicola Bréb

Summary The kinetics of Cu uptake in nutritionally starved cells of the diazotrophic cyanobacteriumNostoc calcicola Bréb. have been compared with those in cells recovering from starvation. Unstarved cyanobacterial cells assimilated 97.0 nmol Cu mg^–1 protein within 1 h when incubated in medium containing 40 M Cu. Uptake was markedly inhibited in carbon-starved cells and, to a lesser extent, in cells starved of nitrogen or sulphur. The intracellular concentrations of protein and photopigments were markedly lower in cells starved of carbon, nitrogen, sulphur or phosphorus, whilst that of carbohydrate was lower in cells starved of carbon, sulphur or phosphorus, but almost doubled in cells starved of nitrogen. The ability to assimilate Cu was partially restored in cells after 72 h of recovery from phosphorus or sulphur deprivation, but showed little improvement during recovery from carbon or nitrogen starvation. A possible role of phosphorus in regulating Cu transport and accumulation is discussed.     

Some Microscopical Observations of Functioning Sieve Tubes of Heracleum using Nomarski Optics

The study of isolated phloem in Heracleum has been extendedto intact functioning sieve tubes. Techniques of phloem dissectioncombined with Nomarski interference optics have been developedto permit useful visual observations, photomicrography, andciné photographs of sieve tubes which are apparentlynormal. In these preparations, plastids and organelles calledby us ‘marker particles’ are visible in rapid bouncingmotion, and the state of dispersal of these particles is relatedto the amount of damage done to the preparations. The movement of the marker particles and their subsequent fixationshows that they are apparently attached to or restrained byan invisible network in situ. The network is very sensitiveto disturbance and readily collapses around the sieve platesto form slime plugs upon damage to the sieve element. The markerparticles do not move through the cell nor across sieve platesin mature Heracleum. In young cells cyclosis is observable alongthe periphery and this suggests that a vacuole may then be present.In undamaged mature cells there was evidence neither of a vacuolenor of trans-cellular tubules of any size optically detectable. The motion of the particles was greater than Brownian movementand appeared to be under some physiological control. Their movementprobably indicated the presence of an operating transport phenomenon,either because solution was moving past them in the sieve tubeor because they were themselves attached to a contractile networkactively in pulsatory motion. Nearby companion and parenchymacells showed normal cytoplasmic streaming. Proposed mechanisms of translocation involving cytoplasmic streamingdo not seem to be applicable to the phloem of Heracleum. Themovement of the marker particles seemed to agree best with amechanism of ‘activated’ mass flow.     

Virus-like particles in yeast: isolation and infectivity.

Virus-like particles containing electron dense cores are seen in thin sections of intact and degenerated cells of a thermosensitive (ts) strain of Candida tropicalis. A particulate fraction not present in wild-type cells has been isolated from the ts cells disrupted by pressure. The particles are 80-120 nm in diameter. Empty particles with a central cavity are observed. The method of infecting mating pairs of Saccharomyces cerevisiae by partially purified particles is described.     

Thienamycin production by immobilized cells of Streptomyces cattleya in a bubble column

A novel 2.0-L columnar reactor has been developed for the production of thienamycin by cells of Streptomyces cattleya attached to celite particles. Successful immobilization of cells was achieved by operating the column continuously at a high dilution rate during the growth phase. Scanning electron micrographs of the celite particles indicate the involvement of subcellular fibrils in the attachment of cells to the solid surfaces. Reactor operation was divided into two distinct phases-a growth phase and a production phase. The kinetics of attached growth and thienamycin production were found to be strongly influenced by nutrient concentrations. The influences of nutrient concentration on CO(2) production and thienamycin production during both the growth phase and the production phase are discussed.     

Effect of polyamines on mitochondrial F1-ATPase catalyzed reactions

The effect of polyamines on F1-ATPase catalyzed reactions has been studied through the use of submitochondrial particles and F1-ATPase. ATP degradation catalyzed by submitochondrial particles and F1-ATPase was inhibited by spermine and spermidine. Spermine's inhibition was much greater than spermidine's effect. In contrast, P1-ATP exchange and succinate dependent ATP synthesis catalyzed by submitochondrial particles were both stimulated by spermine. The inhibition of ATPase activity by polyamines probably occurs through polyamine's replacement of Mg2+ on ATP, for the following reasons. (a) The ATPase activity inhibited by spermine was partially recovered when Mg2+ was added. (b) Spermine bound to ATP and phospholipids but not to F1-ATPase; yet spermine inhibited the ATPase reaction catalyzed by F1-ATPase, a protein free of phospholipid. (c) The binding of spermine to ATP was inhibited by Mg2+. The ATP content in polyamine-deficient cells definitely was lower than that in normal cells. On the basis of these results, the possible role of spermine in keeping the ATP concentration at a high level is discussed.     

Morphological changes in the T=3 capsid of Flock House virus during cell entry

We report the identification and characterization of a viral intermediate formed during infection of Drosophila cells with the nodavirus Flock House virus (FHV). We observed that even at a very low multiplicity of infection, only 70% of the input virus stayed attached to or entered the cells, while the remaining 30% of the virus eluted from cells after initial binding. The eluted FHV particles did not rebind to Drosophila cells and, thus, could no longer initiate infection by the receptor-mediated entry pathway. FHV virus-like particles with the same capsid composition as native FHV but containing cellular RNA also exhibited formation of eluted particles when incubated with the cells. A maturation cleavage-defective mutant of FHV, however, did not. Compared to na?ve FHV particles, i.e., particles that had never been incubated with cells, eluted particles showed an acid-sensitive phenotype and morphological alterations. Furthermore, eluted particles had lost a fraction of the internally located capsid protein gamma. Based on these results, we hypothesize that FHV eluted particles represent an infection intermediate analogous to eluted particles observed for members of the family Picornaviridae.     

Fractionation and Estimation of Particle-Attached and Unattached Bradyrhizobium japonicum Strains in Soils

Rhizobial cells attached or unattached to soil particles were estimated. Nonsterile soils into which antibiotic-resistant mutants of Bradyrhizobium japonicum had been introduced were fractionated by a centrifugation technique into two fractions: A, which contained mainly rhizobial cells attached to soil particles, and F, which contained mainly rhizobial cells unattached to them. Rhizobial counts decreased in both fractions during incubation of the soil at 30°C, with a concomitant decrease in the proportion of the count of fraction F to that of fraction A. Sonication of fraction A of the soil incubated for more than 3 weeks caused an increase in the rhizobial count. The ratio of the count of fraction A estimated by the plant infection method to that estimated by the dilution plate method increased after 5 days of soil incubation. More than 90% of the indigenous rhizobia in an agricultural field existed in fraction A. These results suggest that the majority of rhizobial cells are attached to soil particles.     

Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation.

Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to the film surface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration.