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[Protein traffic in Plasmodium infected-red blood cells].

To survive within erythrocytes, Plasmodium parasites have to put into place different membrane and sub-cellular compartments in order to import different nutrients and to export proteins/antigens. Infected cells pose not only a major world health risk by killing two million people per year, but also a very interesting cell biology problem, as within the erythrocyte the parasite resides inside a vacuole called the parasitophorous vacuole and as a consequence, it is separated from the blood stream by three membrane barriers, its own plasma membrane, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In spite of these three barriers the parasite is capable of secreting antigens and importing nutrients, and to do this, it has developed a complex vesicular system that extends into the red blood cell cytoplasm to the plasma membrane. Understanding how the parasite controls this extensive vesicular traffic has driven research into Plasmodium Rabs, whose potential role is discussed.

Animals↗

Subacute skeletal myopathy induced by 2,4-dichlorophenoxyacetate in rats and guinea pigs.

Rats and guinea pigs were injected with 2,4-dichlorophenoxyacetate (2,4-D) in amounts sufficient to keep them myotonic for over 12 hr daily. The biceps brachii muscles were studied by histochemistry, epoxy resin histology, and electron microscopy after one to five days. Changes observed include proliferation of the longitudinal component of the sarcoplasmic reticulum, production of new myofilaments on the periphery of cells and in nuclei, activation of satellite cells, accumulation of lipid droplets, myofibrillar damage with Z-disc streaming, necrosis, and regeneration. The action mehanisms of 2,4-D are not well understood. Some of the phenomena observed could be classified as proliferative and may be analogous to the auxin action of 2,4-D in plants. The production of intranuclear filaments is probably related to the diffusion of newly formed cytoplasmic proteins into nuclei.

2,4-Dichlorophenoxyacetic Acid↗

Melittin-induced membrane permeability: a nonosmotic mechanism of cell death.

Derived from honeybees, melittin is a 26-amino acid, alpha-helical, membrane-attack protein that efficiently kills mammalian cells. To investigate the contribution of colloid-osmotic effects to the mechanism of cell death, we studied the effect of melittin on lymphocyte membrane permeability and cell volumes. Melittin concentrations of 0.5 to 2.0 microM induced release of membrane permeability markers without total disruption of the cell membrane. At these melittin concentrations, electrical-impedance cytometry demonstrated melittin-induced changes in red blood cell volumes (P<0.01), but no change in lymphocyte cell volumes (P>0.05). Streaming video microscopy, obtaining images of melittin-treated lymphocytes at 80-ms intervals, demonstrated a loss of optical density (P<0.001) suggesting a flattening of the cell but no significant increase in cell perimeter (P>0.05). Real-time multiparameter flow cytometry of melittin-treated lymphocytes confirmed simultaneous loss of the cytoplasmic marker, calcein, and uptake of the DNA dye, ethidium homodimer, but demonstrated no increase in forward light scatter. Transmission-electron microscopy of melittin-treated lymphocytes showed normal cell volumes but discontinuities in the cell membrane suggesting direct membrane toxicity. We conclude that melittin causes lymphocyte death by a "leaky patch" mechanism that is independent of colloid-osmotic effects.

Animals↗

[Immunotyping of medullary and circulatory cells for prognostic evaluation of plasmacytoma].

The authors discuss the prognostic impact of immunophenotyping of circulating lymphoplasmatic cells in the peripheral blood stream in patients with generalized plasmocytoma. From a group of 250 patients followed up from 1981 to 1991 they selected a sub-group of 70 patients where they evaluated in 1986-1991 after six-month intervals the phenotype of medullary and circulating cells. They used the method of immunofluorescent detection of the presence of cytoplasmic Ig, the kappa-lambda index and phenotyping of antigens CD 9, CD 10, CD 20, CD 38, HLA-DR by monoclonal antibodies. In a longitudinal investigation of the survival period they revealed that the finding of circulating cells with signs of non-differentiation (presence of antigen CD 10 detected by antibody CALLA, presence of antigens B 1 (CD 20), CD 9 on circulating lymphocytes) has a prognostic meaning suggesting shorter survival. There was a direct correlation between the increase of CALLA positive cells and CD 9 positive cells. The authors found also that release of the clonus with signs of immaturity was present when the disease developed into the aggressive stage. While the group of 250 patients had according to statistical analyses, when treated according to protocol VMCP/MOCCA, a median survival of 90 months, the median survival of the aggressive stage (with the plasmoblast and lymphoplasmocytic type resp.) was only 12 months. The authors emphasize the prognostic importance of immunological typing of heterogeneous plasmocytoma populations.

Adult↗

Subependymal zone: immunohistochemically distinct compartment in the adult mammalian forebrain.

The subependymal zone (SEZ) lining lateral walls of the lateral cerebral ventricles represents the site of active neurogenesis in the brain of adult mammals. Peroxidase immunohistochemistry performed in paraffin-embedded sections reveals that structural organization of the SEZ differs from other regions in the brain. The SEZ is devoid of synapses that are abundant in the adjacent striatal neuropil. Therefore immunostaining of synaptophysin detects sharp borders of the SEZ. Using immunophenotypization, we identified cell types constituting the SEZ in the intact rat forebrain. The presence of neural progenitor/stem cells was confirmed by finding of nestin-immunopositive cells. Detection of the astroglial marker GFAP confirmed that astrocytes represented major supporting elements responsible for creating a unique microenvironment of the SEZ. One type of the astroglia participated in covering surfaces of the blood vessels and boundaries of the SEZ. The second astroglial cell type formed branched elongated tubes that enwrapped other SEZ cell types with their cytoplasmic extensions. The interior of astrocytic channels was occupied with small densely aggregated NCAM-immunoreactive neuroblasts. Bipolar morphology indicated that these cells probably underwent migration. Immunodetection of other neuronal markers like beta-III tubulin, MAP-2 and Pan neurofilaments identified positive cells in the neighbouring brain parenchyma but not in the SEZ. The rostral migratory stream (RMS) linked with the anterior SEZ had a similar structural arrangement. It contained a large amount of nestin+ and vimentin+ cells. The RMS consisted of GFAP+ astrocytic tubes ensheathing NCAM+ neuroblasts. On the contrary to the SEZ, the RMS neuroblasts expressed beta-III tubulin. However, markers of postmitotic neurons MAP-2, Pan neurofilaments and synaptophysin were not expressed in the RMS. Our study describes a complex histological structure of the rat SEZ, identifies its individual cell types and demonstrates a usefulness of immunohistochemical detection of cell-specific markers in a study of microenvironment forming neurogenic zones in the mammalian brain.

Animals↗

Observations on cell growth, mitosis, and division in the fungus Basidiobolus ranarum.

The paper describes the forward streaming, growth, and division of the vegetative cell of Basidiobolus ranarum. The cell is several hundred microns long and has a single large nucleus. Mitosis is invariably followed by cell division. Both processes have been studied in the living cell by ordinary and phase contrast microscopy. Mitosis is accompanied by a temporary coarsening of the organisation of the cytoplasm and a considerable slowing down of the rate of growth of the cell wall tube. Fixed and stained preparations have shown that there is a large number of small chromosomes and that the mitotic spindle is formed from the nucleolus. Basidiobolus appears suitable for observations on the cell duplication cycle and the physiology of mitosis.

Cell Cycle↗

Cellular and subcellular distribution of HNK-1 immunoreactivity in the neural tube of Xenopus.

The HNK-1 antigen, a carbohydrate moiety bound to many cell adhesion and recognition molecules, is implicated in cell-cell and cell-substrate interactions during neural development. HNK-1 immunoreactivity (HNK1-IR) appears on neurons of the Xenopus neural tube very early in their development (Nordlander, Devel. Brain Res., 50:147-153, 1989). The distribution and onset of expression of the HNK-1 epitope on and within individual neurons is examined in this study. HNK-1 labels developing neurons and their processes, and focal areas of other structures which are directly contacted by neurons, such as neuroepithelial cell surfaces, basal lamina, and culture surfaces. HNK1-IR first appears in the Golgi apparatus and subsequently on the cell surface and in streams of punctate material directed toward the site of axon initiation and into the developing axon and its growth cone. The entire neuron is coated with a thin (20-30 nm) surface layer of HNK1-IR. In addition, the surface is dotted with small (100-250 nm) boluses of HNK1-IR material. Such boluses also occur within cytoplasmic vesicles, and extracellularly on basal lamina and culture substrata in proximity to neurons or their processes. The subcellar distribution of HNK1-IR in this tissue is compatible with a role for the HNK-1 epitope in axonal outgrowth and guidance.

Animals↗

Thyroid hormone receptor monomer, homodimer, and heterodimer (with retinoid-X receptor) contact different nucleotide sequences in thyroid hormone response elements.

Thyroid hormone receptors (TRs) bind as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXRs) to thyroid hormone response elements (TREs). However, it is not known which nucleotides each TR complex contacts in a particular TRE. To identify the precise contact sites on a synthetic DR4 (TRE half-sites arranged as a direct repeat with a four-nucleotide spacer) and a chick lysoenzyme TRE, F2 (half-sites arranged as an inverted palindrome with a six-nucleotide spacer), for various TR complexes, mobility shift assays, and dimethylsulfate and KMnO4 DNA modification interference assays were employed. First, TR alpha monomer bound to the downstream half-site of these TREs, whereas TR alpha homodimer bound to both half-sites. TR alpha/RXR alpha heterodimer also bound to both half-sites, but "preferred" to contact the down-stream half-site. Second, the specific flanking and spacing sequences of DR4 influenced the contact sites and the binding of TR alpha monomer and homodimer, but not TR alpha/RXR alpha heterodimer. Finally, cotransfection studies, using reporter plasmids containing DR4 or F2 in both orientations with respect to the basal promoter, provide evidence that preferential contact with the down-stream half-site by TR/RXR heterodimer may be important for maximal transcriptional activation.

Animals↗

Synchrony in the rhythm of the contraction-relaxation cycle in two plasmodial strands of Physarum polycephalum.

Rhythmicity of contraction of plasmodial strands of Physarum polycephalum was studied, by measuring the isometric tension exerted by isolated segments of the strands. When 2 strands were connected by way of a plasmodial mass, the contraction-relaxation cycle of the 2 strands synchronized. Such cycle activity of the strand was found to be well coordinated with shuttle streaming in the plasmodial mass which had been in connection with the strand. The presence of a control system which induces synchrony of periodic activity over the entire body of a plasmodium is discussed.

Biomechanical Phenomena↗

Reactivation of organelle movements along the cytoskeletal framework of a giant freshwater ameba.

The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments.

Actin Cytoskeleton↗

An electron microscopic study of the intestinal villus. II. The pathway of fat absorption.

The intestinal pathway for absorbed fat was traced in thin sections of intestinal villi from rats fed corn oil by stomach tube after a fast of 24 to 40 hours. For electron microscopy the tissues were fixed in chilled buffered osmium tetroxide and embedded in methacrylate. For light microscopy, other specimens from the same animals were fixed in formal-calcium, mordanted in K(2)Cr(2)O(7), and embedded in gelatin. Frozen sections were stained with Sudan black B or Sudan IV. About 20 minutes after feeding, small fat droplets (65 mmicro maximal diameter) appear in the striated border between microvilli. At the same time fat particles are seen within pinocytotic vesicles in the immediately subjacent terminal web. In later specimens the fat droplets are generally larger (50 to 240 mmicro) and lie deeper in the apical cytoplasm. All intracellular fat droplets are loosely enveloped in a thin membrane, the outer surface of which is sometimes studded with the fine particulate component of the cytoplasm. This envelope, apparently derived from the cell surface by pinocytosis, has at this stage evidently become a part of the endoplasmic reticulum. Just above the nucleus numerous fat droplets lie clustered within the dilated cisternae of the Golgi complex. As absorption progresses fat droplets appear in the intercellular spaces of the epithelium, in the interstitial connective tissue spaces of the lamina propria, and in the lumen of the lacteals. All of these extracellular fat droplets are devoid of a membranous envelope. The picture of fat absorption as reconstructed from these studies involves a stream of fat droplets filtering through the striated border, entering the epithelial cell by pinocytosis at the bases of the intermicrovillous spaces, and coursing through the endoplasmic reticulum to be discharged at the sides of the epithelial cell into extracellular spaces. From the epithelial spaces, the droplets move into the lamina propria and thence into the lymph. If the lumen of the endoplasmic reticulum is considered as continuous with the extracellular phase, then the entire pathway of fat absorption may be regarded as extracellular. However, it is impossible to evaluate from the electron microscopic evidence thus far available the quantitative importance of particulate fat absorption by the mechanism described.

Animals↗

Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

Diffusion↗

Cyclic changes in the pituitary gland in correlation with the testicular cycle in a hill-stream teleost, Schizothorax richardsonii (Gray and Hard).

In Schizothorax richardsonii, the pituitary gland has been studied in correlation with the phases of the testicular cycle. The cyanophils of the proximal pars distalis show hypertrophy, hyperplasia, granulation, degranulation and vacuolisation in concurrence with the spermatogenic cycle. Secretory activity of the cyanophils controls the maturation of testes, which indicates that these cyanophils are gonadotrops. The formation of sperm mother cells, however, does not appear to depend on the secretory activity of the pituitary. During spawning and regression phases, the azocarminophilous granules in the cytoplasm of the cyanophils show quantitative variation. The migratory chromophil cells show numerical variation, suggesting their involvement in the reproductive cycle. Neurosecretory material also appears to play some part in reproduction.

Animals↗

Ultrastructural features of the endomyocardium in patients with eosinophilic heart disease. An endomyocardial biopsy study.

In a series of studies with endomyocardial biopsy, 7 adult cases with cardiac disease and eosinophilia were studied clinically and electro-microscopy. Degranulation of the eosinophils in the peripheral blood was observed ultrastructurally in 3 of the 4 cases studied. The clinical expression of the 7 patients were, restrictive cardiomyopathy in 2, dilated cardiomyopathy in 2 and sick sinus syndrome in 1 and others in 2. Endocardial thickening was observed in 5 cases, one of whom showed a marked cellular infiltration with macrophages, plasma cells, lymphocytes and mast cells. One other case showed cell debris of degranulated eosinophils. Degeneration of the myocytes was manifested by an increase in Z-bands of the myofibrils as well as streaming (1 case), disarrangement of the myofibrils, and mitochondrial change (1 case) which was characterized by giant mitochondria (1 case) as well as by a numerical increase in mitochondria. Increase of atrial granules and mitochondria in a right atrial biopsy of a case with sick sinus syndrome was noteworthy. Basal lamina layering of the capillaries of the myocardium and pyknosis of an endothelial cell cytoplasm were also noted in each case. These observations may reveal that various disease processes are taking place in the endocardium and adjacent myocardium.

Adult↗

Rheological properties of living cytoplasm: endoplasm of Physarum plasmodium.

Magnetic sphere viscoelastometry, video microscopy, and the Kamiya double chamber method (Kamiya, N., 1940, Science [Wash. DC], 92:462-463.) have been combined in an optical and rheological investigation of the living endoplasm of Physarum polycephalum. The rheological properties examined were yield stress, viscosity (as a function of shear), and elasticity. These parameters were evaluated in directions perpendicular; (X) and parallel (Y) to the plasmodial vein. Known magnetic forces were used for measurements in the X direction, while the falling ball technique was used in the Y direction (Cygan, D.A., and B. Caswell, 1971, Trans. Soc. Rheol. 15:663-683; MacLean-Fletcher, S.D., and T.D. Pollard, 1980, J. Cell Biol., 85:414-428). Approximate yield stresses were calculated in the X and Y directions of 0.58 and 1.05 dyn/cm2, respectively. Apparent viscosities measured in the two directions (eta x and eta y) were found to fluctuate with time. The fluctuations in eta x and eta y were shown, statistically, to occur independently of each other. Frequency correlation with dynamoplasmograms indicated that these fluctuations probably occur independently of the streaming cycle. Viscosity was found to be a complex function of shear, indicating that the endoplasm is non-Newtonian. Plots of shear stress vs. rate of shear both parallel and perpendicular to the vein, showed that endoplasm is not a shear thinning material. These experiments have shown that living endoplasm of Physarum is an anisotropic viscoelastic fluid with a yield stress. The endoplasm appears not to be a homogeneous material, but to be composed of heterogeneous domains.

Cytoplasm↗

Relations between ameboid movement and membrane-controlled electrical currents.

We have studied the pattern of electrical currents through amebas (mainly Chaos chaos) with an ultrasensitive extracellular vibrating probe. Amebas drive both steady currents and current pulses through themselves. Relatively steady current with an average surface density of 0.1-0.2 muA/cm2 enters the rear quarter of an ameba and leaves its pseudopods. Streaming reversals are preceded by changes in this current pattern and the region with the largest new inward current becomes the new tail. Ion substitution studies suggest that some of the steady inward current is carried by calcium ions. Characteristic stimulated pulses of current sometimes follow the close approach of the vibrating probe to the side of an advancing pseudopod. Such a pulse enters the cytoplasm through a small patch of membrane near the probe (and seems to leave through the adjacent membrane), is usually followed by hyaline cap and then by pseudopod initiation, is calcium dependent, lasts about 5-10 s, and has a peak density of about 0.4 muA/cm2. Spontaneous pulses of similar shape and duration may enter or leave any part of an animal. They are much less localized, tend to have higher peak densities, and occur in physiological salt solutions at about 0.2-4 times per minute. Retraction of a pseudopod is always accompanied or preceded by a spontaneous pulse which leaves its sides.

Amoeba↗

Reduction of mercury to the elemental state by a yeast.

A yeast of the genus Cryptococcus has been isolated from a stream and was shown to be capable of reducing mercury to the elemental state. The organism grows in Wickerham broth supplemented with high concentrations of mercury (II) chloride (180 mg of mercury per liter) and will metabolize [(14)C]glucose in this medium as do cells in the absence of mercury. Mercury was associated with the cell wall and membrane, and in vacuoles within the cytoplasm.

Carbon Dioxide↗

Lymph macrophages enter the germinal center of lymph nodes of guinea pigs.

To determine the fate of macrophages within the afferent lymph stream, the popliteal lymph nodes at various times (3 h to 6 months) after subcutaneous injection of india ink into the footpads of guinea pigs were examined. Two types of cells which had phagocytized india ink were observed in the germinal centers. A small number of type I phagocytes (engulfing india ink as small particles which were often found together with tingible bodies in their cytoplasm) were scattered through the germinal center. A large number of type II phagocytes (packed full of india ink) presented preferentially in the medullary portion of the germinal center, together with many pyroninophil lymphoblastoid cells. In the second experiment, the afferent lymphatics of the popliteal lymph node were ligated 15-20 min after india ink injection. Although the type I phagocytes were distributed as in the first experiment, the type II phagocytes were scanty. In the third experiment, the afferent lymphatics of the popliteal lymph node were ligated 7 days after india ink injection. The type II phagocytes disappeared rapidly from the germinal center, whereas the type I phagocytes remained and were not affected by ligature. These results suggest that the type I phagocytes are the fixed macrophages or tingible body macrophages in the germinal center, and that the type II phagocytes are the macrophages migrating from the peripheral tissues. It was also shown that many macrophages reaching the regional node via afferent lymphatics entered the germinal center through the medullary pole where the cap of small lymphocytes became thinner or disappeared.

Animals↗