Effect of enthidium on the morphology, antiviral activity and subcellular distribution of Poly r(A-U)

When ethidium bromide (EB) is combined with poly r(A-U) at an EB/ribonucleotide ratio of 1/4, the antiviral activity of the EB increases 22-fold. The increased antiviral activity is not due to increased interferon induction, direct viral inactivation or host cell cytotoxicity. Phase contrast, confocal and fluorescence microscopic observations reveal an increase in the nucleolar accumulation of the EB and/or the poly r(A-U) in the EB/poly r(A-U)-treated fibroblasts. Ultrastructure of negatively stained and replica preparations demonstrated that EB-induced condensation of poly r(A-U). These results suggest the elevated antiviral activity may be related to the altered uptake and subcellular distribution of the EB/poly r(A-U) complex.     

Mechano-electrical vibrations of microtubules-Link to subcellular morphology

Spontaneous mechanical oscillations were predicted and experimentally proven on almost every level of cellular structure. Besides morphogenetic potential of oscillatory mechanical force, oscillations may drive vibrations of electrically polar structures or these structures themselves may oscillate on their own natural frequencies. Vibrations of electric charge will generate oscillating electric field, role of which in morphogenesis is discussed in this paper. This idea is demonstrated in silico on the conformation of two growing microtubules.     

Effect of UV-irradiation on DNA replication of the parvovirus minute-virus-of-mice in mouse fibroblasts.

The effect of UV-irradiation on the conversion of the single-stranded DNA of the parvovirus Minute-Virus-of-Mice (MVM) to duplex Replicative Forms (RF) was studied after infection of mouse A9 fibroblasts. UV-irradiation of the virus prior to infection of unirradiated cells resulted in a dose-dependent, single-hit, inhibition of RF formation. Restriction fragment analysis indicated that this inhibition could be ascribed to the introduction of absolute blocks which prevent elongation of the newly synthesized complementary strand. Cell exposure to UV-light prior to infection with UV-irradiated MVM enhanced the fraction of input viral DNA which was converted to RF. This enhancement required de novo protein synthesis during the interval between cell irradiation and virus infection. These results suggest that DNA replication constitutes a target in the viral life cycle that leads to the UV-enhanced Reactivation of virus survival, however, they do not permit us to identify the step of RF formation which is enhanced in UV-pretreated cells.     

Disturbance of the secretory pathway inMicrasterias denticulata by tunicamycin and cyclopiazonic acid

Summary Both tunicamycin, an inhibitor of N-linked glycosylation of proteins, and cyclopiazonic acid, which inhibits the Ca²⁺-dependent ATPase in the ER, influence the secretory pathway at the ER level and lead to a cessation of cell growth inMicrasterias. Electron microscopical investigations reveal that the mode of action of the two inhibitors differs. While tunicamycin treatment results in a disintegration of the Golgi bodies into small vesicles, cyclopiazonic acid prevents products being supplied from the ER, resulting in the dilatation of ER cisternae and a reduction in the number of Golgi cisternae, combined with a loss of dictyosomal activity. The disturbed cell wall formation under tunicamycin indicates that N-linked glycosylation of proteins is required for normal cell growth inMicrasterias. Moreover, our studies reveal that changes in cytoplasmic free calcium concentration, as a consequence of ATPase inhibition in the ER by cyclopiazonic acid, may inhibit wall material secretion by interrupting the normal ER-dictyosome association.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Effect of calcium on subcellular distribution of peroxidases

About 40% of the peroxidase activity extracted from hypocotyl hooks of etiolated Cucurbita pepo was pelletable at 20000 g. The activity in the pellet was partially solubilized by the addition of 5 mM EGTA. This effect of EGTA was reversed by Ca²⁺, but not by Mg²⁺. Reassociation of EGTA-solubilized peroxidases to 4 cellular fractions obtained by centrifugation on discontinuous sucrose gradients was assayed. It appeared that the enzymes could be linked to ribosomes or RNP particles through Ca²⁺. Two fractions, identified as smooth and rough endoplasmic reticulum, also bound peroxidases and, in this case, the Ca²⁺-mediated binding involved a loss of the enzyme activity. The fraction containing mitochondria and plasmalemma exhibited a slight binding capacity. Isoelectric focusing in thin layer polyacrylamide gel showed that only 5 out of the 10 isoperoxidases present in hypocotyl hooks had their pelletability level changed by Ca²⁺.     

Effect of hypoxia on the binding and subcellular distribution of iron regulatory proteins

Iron regulatory proteins 1 and 2 (IRP1, IRP2) are key determinants of uptake and storage of iron by the liver, and are responsive to oxidative stress and hypoxia potentially at the level of both protein concentration and mRNA-binding activity. We examined the effect of hypoxia (1% O₂) on IRP1 and IRP2 levels (Western blots) and mRNA-binding activity (gel shift assays) in human hepatoma HepG2 cells, and compared them with HEK 293 cells, a renal cell line known to respond to hypoxia. Total IRP binding to an iron responsive element (IRE) mRNA probe was increased several fold by hypoxia in HEK 293 cells, maximally at 4–8 h. An earlier and more modest increase (1.5- to 2-fold, peaking at 2 h and then declining) was seen in HepG2 cells. In both cell lines, IRP1 made a greater contribution to IRE-binding activity than IRP2. IRP1 protein levels were increased slightly by hypoxia in HEK 293 but not in HepG2 cells. IRP1 was distributed between cytosolic and membrane-bound fractions, and in both cells hypoxia increased both the amount and IRE-binding activity of the membrane-associated IRP1 fraction. Further density gradient fractionation of HepG2 membranes revealed that hypoxia caused an increase in total membrane IRP1, with a shift in the membrane-bound fraction from Golgi to an endoplasmic reticulum (ER)-enriched fraction. Translocation of IRP to the ER has previously been shown to stabilize transferrin receptor mRNA, thus increasing iron availability to the cell. Iron depletion with deferoxamine also caused an increase in ER-associated IRP1. Phorbol ester caused serine phosphorylation of IRP1 and increased its association with the ER. The calcium ionophore ionomycin likewise increased ER-associated IRP1, without affecting total IRE-binding activity. We conclude that IRP1 is translocated to the ER by multiple signals in HepG2 cells, including hypoxia, thereby facilitating its role in regulation of hepatic gene expression. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.     

Effect of digitonin on rat myometrium subcellular membrane fractions

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.     

Effect of penicillin on the morphology and reproduction ofAzotobacter chroococcum

The effect of penicillin on the morphology and reproduction of some strains ofA. chroococcum was studied on a number of solid media. When the growth was not entirely suppressed by the penicillin, filamentous cells and spheroplasts were formed. The formation of spheroplasts was stimulated by peptone. Gonidia were sometimes formed inside the spheroplasts and also inside giant cells. They were released from the cell after disruption or after lysis of the cell wall. In some cases they produced dwarf cells. Under certain conditions groups of gonidia present in a cell fused and formed one or more normal-looking cells inside the mother cell. Sometimes one or moreAzotobacter cells developed inside a spheroplast or at the site of a spheroplast with a lysed cell wall. Microcolonies consisting of small cocci representing gonidia and dwarf cells were also observed occasionally at the sites of spheroplasts with lysed cell walls. Occasionally tiny groups of small elements with a less marked structure were found at such sites, probably representing debris of lysed cells. The production of normal-looking cells inside filamentous cells was greatly stimulated on a medium containing 10 percent horse serum, with a drop of sterile water containing 200 or 250 I.U. penicillin added in the centre of the plate. The growth ofA. chroococcum was greatly retarded when the medium contained 10 U/ml penicillin and seemed to be checked entirely at concentrations of 20 U/ml penicillin or higher. Occasionally, however, even at concentrations of 100 and 300 U/ml penicillin, a few filamentous cells were found and also a few microcolonies, visible only through the microscope, consisting of gonidia or regenerative rods. By repeated exposure ofAzotobacter to penicillin populations could be obtained that were adapted to high concentrations of this antibiotic.     

Effects of UV-irradiation on the SCE frequency in human lymphocyte cultures

UV-irradiation (254 nm) was found to induce a smaller increase of SCE in human lymphocytes than in human fibroblasts and CHO cells. The UV-induced SCE frequency in human lymphocytes was not influenced by the duration between irradiation and the subsequent S-phase. UV-irradiated lymphocytes showed a slightly more than additive response to the SCE-inducing effect of HN2 and acetaldehyde in comparison with non-irradiated cells. The UV-induced SCE frequency was similar in lymphocyte cultures containing 20 and 100 microM of BrdUrd. The results suggest that human lymphocytes are relatively insensitive to the SCE-inducing effect of UV-irradiation, and that SCE-inducing damage caused by UV is not removed during the G1 phase in these cells.     

Effects of whole body UV-irradiation on oxygen delivery from the erythrocyte

In 16 healthy caucasian volunteers (mean age: 22.2 years) the influence of whole body UV-irradiation on the oxygen transport properties of erythrocytes was investigated. Four hours after irradiation with UV (using the minimal erythema dose, MED) no variation of haemoglobin concentration, hematocrit, mean corpuscular haemoglobin concentration, pH or standard bicarbonate could be found, whereas inorganic plasma phosphate (Pi), calcium, the intraerythrocytic 2,3-diphosphoglycerate (2,3-DPG), the activity of erythrocytic phosphofructokinase (PFK) and pyruvatekinase (PK) increased significantly. The half saturation tension of oxygen (P50-value) tended to increase. The increase of Pi causes--via a stimulation of the glycolytic pathway--an increase in 2,3-DPG concentration and thus results in a shift of the oxygen dissociation curve. It is therefore possible to enhance tissue oxygenation by whole body UV-irradiation.     

Effect of lipopolysaccharides on the morphology of erythrocyte membranes

The effect of lipopolysaccharide (LPS) isolated from Gram-negative bacteriaSalmonella typhi on erythrocyte membranes was investigated by electron microscopy. The LPS was found to be irregularly distributed on the surface of erythrocytes and their envelopes. It caused the formation of short bilayer rods associated at one end with the erythrocyte membrane of vesicular formations localized horizontally on or in the membrane, and of lamellar structures.     

Effect of UV-irradiation, spleen cells and MLN cells on protective immunity against Angiostrongylus cantonensis infection in mice

The UV-irradiated larvae of Angiostrongylus cantonensis were used for inducing immunity in mice. A single oral dose of 100 infective UV-irradiated larvae exposed for 5 min or 15 min induced 91% and 97% protection against subsequent infection. Adoptive protection against A. cantonensis could also be transferred by spleen and mesenteric lymph node cells obtained from vaccinated mice.     

Effect of hydration on the morphology of enzyme powder

We report the first direct images of the hydration of protein powders. Using an environmental scanning electron microscope (ESEM) we have taken a series of micrographs of a region of the enzyme (subtilisin) power whilst hydrating the sample. In addition, the sample has been viewed during exposure to toluene vapors. The ESEM is a remarkable new instrument that will have wide applicability in imaging of biological materials in their native environments.     

Effect of ischemia-reperfusion injury on the morphology of peroxisomes

We have previously demonstrated that ischemic injury changed the density of peroxisomes into two distinct peaks, one with a normal density (1.21 g/cm³; Peak I) and a second peak with a lighter density (1. 14 g/cm³; Peak II).We studied the peroxisomes from both peaks under the Electron microscope. Examination of peak I following ischemia showed loss of matrix proteins and damaged limiting membranes with leakage of DAB positive material in direct proportion to the duration of ischemia. Upon reperfusion of the ischemic liver Peak I showed more severe damage to the organelle. These observations clearly demonstrated that ischemia reperfusion injury causes structural damage to peroxisomes. Interestingly ultrastructural examination of Peak II following ischemia showed evidence of perisomal proliferation with budding of existing peroxisomes and the presence of micro peroxisomes (changes similar to those noted under conditions leading to perisomal proliferation). However, peak II following reperfusion showed only damaged organelle. These observations underline the importance of peroxisomes in the response of the cell to ischemia-reperfusion injury.