Quantitative ultrastructural study of nucleolus-organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis)

The quantitative characteristics of chromosomal nucleolus-organizing regions (NORs) and some other nucleolar components were studied on ultra-thin sections of pig embryo kidney cells (PK cells). It was shown that: 1) nucleoli-per-cell volumes were 3 times smaller in the G0 period than in the G2 period; 2) the number of fibrillar centers (FCs) per cell in the G0 period, the G2 period, and at metaphase was equal to 7, 33.7, and 8, respectively; 3) mean volumes of individual FCs in the G0 period (0.033 +/- 0.005 micron3), G2 period (0.014 +/- 0.001 micron3), and at metaphase (0.025 +/- 0.002 micron3) were significantly different; 4) the total volumes of FCs calculated per haploid set of chromosomes were practically the same in the G0 (0.105 micron3) and G2 (0.107 micron3) periods, but were twice as large as those at metaphase (0.04-0.05 micron3). These data show that partial activation and inactivation of ribosomal genes in interphase PK cells are not accompanied by a considerable change in the total volume of FCs and may be due to the fragmentation and fusion of individual FCs. Complete inactivation of ribosomal genes in mitosis results in a decrease of total volumes of FCs per cell; 5) in G0 and G2 periods the total volume of the dense fibrillar component per nucleolus is practically proportional to the nucleolus volume (r = 0.99); 6) in the G2 period, the nucleolus volume is also proportional to the number of FCs (r = 0.99; 7) the volume of the dense fibrillar component within individual fibrillar complexes is not a constant one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-derived growth factor: morphologic and biochemical studies of binding, internalization, and degradation

Kinetic studies of binding and internalization of 125I-platelet-derived growth factor (PDGF) demonstrate that up to 15% of membrane-associated radioactivity is internalized within 2 minutes after warming to 37 degrees C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60-90 minutes following initiation of internalization. Internalization and lysosomal association of 125I-PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold-PDGF) demonstrate that 17% of the cell-associated sites are along coated regions of the plasma membrane (1.0 sites/micron), while 82% are associated with noncoated membrane (0.2 sites/micron). There is a significant redistribution of the gold-PDGF complexes upon warming. Within 1-2 minutes at 37 degrees C, gold particles are found within endocytic vesicles, endosomes (0.09-0.3 micron diameter), and lysosomes (greater than 0.2 micron diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per micron2 of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per micron2 of surface area. Simultaneously, there is an increase in the number of gold-PDGF binding sites within coated-pits (1.6 sites/micron, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/micron, 58% of the total sites). After 15 minutes at 37 degrees C, 26% of the total sites (1.4 sites/micron2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/micron, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold-PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold-PDGF is processed via both receptor-mediated and nonspecific endocytosis and follows an intracellular pathway comparable to that followed by some other protein ligands.     

Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

Brain chromosomal DNA isolated from fetal BDIX-rats 1 h after i.v. administration of the ethylating N-nitroso carcinogen N-ethyl-N-nitrosourea (75 micrograms/g body weight), statistically contained one molecule of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) per 81 micron of DNA, as determined in enzymatic DNA hydrolysates by competitive radio-immunoassay using a high-affinity anti-(O6-EtdGuo) monoclonal antibody (ER-6). After fragmentation of the DNA by the restriction enzyme AluI (average fragment length, Lav = 0.28 micron = 970 bp; length range, Lr = 1.87-0.02 micron = 6540 - 60 bp), a small (approximately 2%) fraction of DNA enriched in specific polypeptides tightly associated with DNA was separated from the bulk DNA by a glass fiber binding technique. As analyzed by immune electron microscopy, approximately 1% of the DNA molecules in this fraction contained clusters of 2-10 (O6-EtdGuo)-antibody binding sites (ABS). On the cluster-bearing fragments (Lav, 0.85 micron +/- 0.50 micron S.D.; corresponding to 2970 +/- 1760 bp) the average ABS-ABS interspace distance was 110 nm (= 390 bp; range approximately 9-600 nm), indicating a highly non-random distribution of O6-EtdGuo in target cell DNA.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

Morphometric study of nucleolus-organizing regions of chromosomes from swine embryo kidney cells during Go-, G2-period and mitosis

Quantitative characteristics of nucleolus-organizing regions of chromosomes (NORs, or fibrillar centers, FCs) and some other nucleolar components have been studied with the aid of complete series of ultrathin sections of PK-cells. It has been found that: 1) the number of FCs per cell in the G0-period, in the G2-period and at metaphase is equal to 7.0, 33.7 and 8.0, respectively; 2) volumes of individual FCs in the G0-period (0.033 micron 3), G2-period (0.014 micron 3) and at metaphase (0.025 micron 3) are different; 3) the total volume of FCs, calculated for a haploid set of chromosomes, do not differ in the G0 (0.105 micron 3) and G2 (0.107 micron 3) periods, but exceed twice the FCs volume at metaphase (0.04-0.05 micron 3). These data show that the activation and inactivation of ribosomal genes in interphase PK-cells are not accompanied by a change in the total volumes of FCs and are probably connected with the "fragmentation" and fusion of FCs. Complete inactivation of ribosomal genes at mitosis leads to a decrease in the total volumes of FCs; 4) the nucleolus volume is proportional to the volume of the dense fibrillar RNP-component; in the G2-period the nucleolus volume also correlates with the number of FCs (r = 0.99); 5) the volume of the dense fibrillar component within individual fibrillar complexes--the structures corresponding to one nucleolus-organizing region--is not constant. This is an indirect evidence for the differences in the functional activity of NORs of different chromosomes.     

Role of the membrane cortex in neutrophil deformation in small pipets.

The simplest model for a neutrophil in its "passive" state views the cell as consisting of a liquid-like cytoplasmic region surrounded by a membrane. The cell surface is in a state of isotropic contraction, which causes the cell to assume a spherical shape. This contraction is characterized by the cortical tension. The cortical tension shows a weak area dilation dependence, and it determines the elastic properties of the cell for small curvature deformations. At high curvature deformations in small pipets (with internal radii less than 1 micron), the measured critical suction pressure for cell flow into the pipet is larger than its estimate from the law of Laplace. A model is proposed where the region consisting of the cytoplasm membrane and the underlying cortex (having a finite thickness) is introduced at the cell surface. The mechanical properties of this region are characterized by the apparent cortical tension (defined as a free contraction energy per unit area) and the apparent bending modulus (introduced as a bending free energy per unit area) of its middle plane. The model predicts that for small curvature deformations (in pipets having radii larger than 1.2 microns) the role of the cortical thickness and the resistance for bending of the membrane-cortex complex is negligible. For high curvature deformations, they lead to elevated suction pressures above the values predicted from the law of Laplace. The existence of elevated suction pressures for pipets with radii from 1 micron down to 0.24 micron is found experimentally. The measured excess suction pressures cannot be explained only by the modified law of Laplace (for a cortex with finite thickness and negligible bending resistance), because it predicts unacceptable high cortical thicknesses (from 0.3 to 0.7 micron). It is concluded that the membrane-cortex complex has an apparent bending modulus from 1 x 10(-18) to 2 x 10(-18) J for a cortex with a thickness from 0.1 micron down to values much smaller than the radius of the smallest pipet (0.24 micron) used in this study.     

Peroxisomes in fibroblasts from skin of Refsum's disease patients

Skin fibroblasts were cultured from young adult patients with Refsum's disease, an inherited metabolic disorder characterized by a deficiency in oxidation of phytanic acid and by increased serum and tissue concentrations of this fatty acid. These cultures were compared to cultures of normal fibroblasts in terms of the number and distribution of peroxisomes demonstrable cytochemically in preparations incubated for catalase activity. Refsum's fibroblasts were found to contain 1-10 peroxisome profiles per 100 micron 2 of cytoplasm; the controls contained 1-2 profiles per 100 micron 2. The peroxisomes in normal fibroblasts were found in all regions of the cytoplasm. In the Refsum's material they were relatively scarce in the perinuclear region, where many of the cells showed numerous large inclusions containing lipid-like material and myelin figures. Our findings indicate that in the adult form of Refsum's disease, which is the more thoroughly studied variety, peroxisomes in fibroblasts are not diminished in number. This contrasts with a recent report concerning a case of what is thought to be an infantile form of the disorder, in which no peroxisomes were detected in a liver biopsy. If phytanic acid accumulations in the adult form are a consequence of peroxisomal defects, the defects presumably are at the level of specific enzymatic deficiencies and do not involve a generalized absence of peroxisomes.     

A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.

The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase.     

An investigation of the presence of ultramicrocells in natural mineral water

The presence of 'ultramicrocells' in natural mineral water, capable of passing through a 0.2 micron filter, has been demonstrated. Filters allowing the greatest proportion of viable (culturable) cells to pass ranked in the order, 0.4 micron polycarbonate (5.02%) > 0.2 micron polycarbonate (0.02%) > or = 0.45 micron cellulose nitrate (0.02%) > 0.2 micron cellulose acetate (< 0.002%). Following incubation for 4 d at 22 degrees C, viable counts in filtered mineral water increased from < 2-8.7 x 10(2) cfu ml-1(-2).8 x 10(4)-1.9 x 10(6) cfu ml-1. Successive filtration/incubation cycles of mineral water increased the proportion of cells passing through a 0.2 micron cellulose acetate filter from < 0.003% to 0.11% and 0.69%, suggesting selection for 'ultramicrocells'. Cells isolated from this process and grown on liquid R2A medium were thin, Gram-negative rods, of 0.15-0.40 micron wide and 0.50-6.20 microns long. Membrane filtration techniques used for pathogen detection in mineral waters will not retain all the cells present. If pathogens are able to form ultramicrocells, these may go undetected.     

Effects of total parenteral nutrition on rat bile canaliculi: a stereologic analysis

OBJECTIVE: To quantitate, in a stereologic manner, changes in bile canalicular morphology before and after choleretic infusion of total parenteral nutrition (TPN) and to determine whether TPN produces changes in localized regions within the hepatic lobule. STUDY DESIGN: Livers were obtained from sham-operated on normal adult male rats (control) and from rats that received intravenous TPN solution containing 20% glucose and 3.5% Molipron F. The tissues, obtained by a rigorous sampling procedure, were systematically subjected to stereologic analysis. Measurements were made on electron micrographs at two levels of magnification by point, intersection and profile counts, and then volume, surface area and length were estimated per unit parenchymal volume. RESULTS: The surface area of the canalicular wall per parenchymal volume increased significantly (from 5.33 x 10(-2) to 6.73 x 10(-2) microns 2/micron 3) after TPN treatment, as did the length of microvilli (from 0.241 to 0.267 microns/micron 3). However, the volume of bile canalicular lumina per parenchymal volume (0.306% and 0.320%), total length of bile canaliculi (1.05 x 10(-2) and 1.06 x 10(-2) microns/micron 3) and diameter of microvilli (8.73 x 10(-2) and 8.94 x 10(-2) microns) remained constant. CONCLUSION: These results indicate that changes in canalicular shape and microvillus hypertrophy may cause lowering efficiency of the bile flow rate.     

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219- 232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).     

Scanning electron microscopy of the vitelline membrane of the hen ovum

Manual removal of the perivitelline layer overlying the animal pole (AP) reveals three morphologically distinct regions of the vitelline membrane (VM). (1) The central germinal region is 600-800 micron in diameter and is densely populated with pleomorphic microvillous projections. (2) The periblastic region, which also exhibits microvillous projections, is 250-550 micron wide and consists of numerous (80-120) lacunae that are 10-60 micron in diameter and up to 20 micron in depth. (3) At the outer periblastic region, the microvillous projections are less numerous. In the vegetal hemisphere, the VM has few projections and occasionally is discontinuous.     

Perinuclear microtubules in postnatal rat heart

Cytoplasmic microtubules can be divided into two subpopulations: 1) those adjacent to the nucleus (perinuclear), and 2) those distributed between the myofilament bundles (nonperinuclear). Previous observations (Cartwright and Goldstein, '83) indicate total cytoplasmic microtubule numeric density increases to a maximum at 5-9 days and decreases to the steady value of the adult muscle. We have examined the numeric density (mean numbers of microtubule profiles per micron2 cross-sectional area) of the perinuclear subpopulation and compared it to the numeric density of the total cytoplasmic microtubule population in postnatally developing rat papillary muscle ages 1, 3, 5, 9, 21, and 42 days, and adult. The perinuclear region was defined as the area around the nucleus which extends to the 0.273 micron from the nuclear envelope. The density of perinuclear microtubules did not change with postnatal development. Our study suggests that perinuclear microtubules are a separate and relatively stable subpopulation of the total population of cytoplasmic microtubules and may serve a function different from that of the more variable nonperinuclear microtubules.     

Quantitative deposition of ultrafine stable particles in the human respiratory tract

Theoretical models of particle deposition in the respiratory tract predict high fractional deposition for particles of less than 0.1 micron, but there are few confirming experimental data for those predictions. We have measured the deposition fraction of a nonhygroscopic aerosol in the human respiratory tract. The aerosol had a count mean diameter of 0.044 micron SD of 1.93, as measured with an electrical aerosol analyzer, and was produced from a 0.01% solution of bis(2-ethylhexyl) sebacate using a condensation generator. Subjects inhaled the aerosol using a controlled respiratory pattern of 1 liter tidal volume, 12/min. Deposition was calculated as the difference in concentration between inhaled and exhaled aerosol of five size fractions corrected for system deposition and dead-space constants. Three deposition studies were done on each of five normal male volunteers. Means (+/- SE) for the five size fractions were 0.024 micron, 0.71 +/- 0.06; 0.043 micron, 0.62 +/- 0.06; 0.075 micron, 0.53 +/- 0.05; 0.13 micron, 0.44 +/- 0.04; and 0.24 micron, 0.37 +/- 0.06. These data demonstrate that deposition of inhaled particles in the 0.024- to 0.24-micron size range is high and increases with decreasing size. These observations agree with and validate predictions of mathematical models.     

Calibration of beam deflection produced by cellular forces in the 10(-9)--10(-6) gram range

An experimental procedure and method of analysis are presented for calibration of a thin-beam force transducer. The beam transducer can be produced and calibrated with a minimum coefficient of 10 ng (10(-5) dyne) force per micron (10(-4) cm) deflection, i.e., kB approximately 0.1 dyne/cm. Since beam deflections on the order of 0.1 micron can be detected, forces of a few nanograms can be resolved. Such forces are common in mechanical experiments on microscopic bodies, e.g., biological cells, artificial membrane capsules, droplets, etc.     

Eimeria waiganiensis sp. n. from the green-winged ground dove (Chalcophaps indica Linnaeus) and the magnificent ground pigeon (Otidiphaps nobilis Gould) in Papua New Guinea

Eimeria waiganiensis sp. n. is described from Green-winged ground dove (Chalcophaps indica Linnaeus) and the Magnificent ground pigeon (Otidiphaps nobilis Gould) in Papua New Guinea. Oocysts broadly ovoid, 22-25 by 19-23 micron (24 by 22 micron); L/W ratio 1.08-1.2 (1.1). Oocyst surface smooth, wall light brown, 1.5 micron thick, 1-layered, the inner surface appearing pitted. Micropyle (4-6 micron) and 2 to 4 polar granules present; oocyst residuum absent. Sporocysts ovoid, 9.0-10.5 by 6.0-7.5 micron (10 by 7 micron) with prominent conical Stieda body and small substieda body. Small, finely-granular sporocyst residuum is sanwiched between the sporozoites. The elongate-ovoid sporozoites possess distinct anterior and posterior refractile globules. Two of 4 ground doves and 1 of 3 ground pigeons were infected with E. waiganiensis sp. n.     

Enrichment of viable lymphocytes in defined cycle phases by sorting on the basis of pulse width of axial light extinction

A correlation between cell size and cycle state is demonstrated for mitogen stimulated murine lymphocytes. Cell cycle analysis was performed using multiparameter cytofluorometric analysis of acridine orange stained cells. Cell size was determined by measuring "time of flight" of axial light extinction. Cells with diameters of 5.0 - 6.0 micron are shown to be predominately in G0. Among stimulated cells, those 6.0 - 7.0 micron in diameter are predominately early and late G1 (80%) with some S phase cells. Cells in G2 + M were found to predominate among lymphocytes with diameters of 7.5 - 12 micron. This approach provides a method for isolation of lymphocytes in specific phases of the cell cycle which are suitable for subsequent functional studies.     

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization.

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.     

On the binding of labelled saxitoxin to the squid giant axon

Measurement of the maximum saturable binding capacity for 3H-labelled saxitoxin in well cleaned giant axons from Loligo forbesi gave a value of 290 +/- 65 (s.e.m.) binding sites per square micrometre of membrane. In samples of the fin nerve the total saturable uptake was 12.4 +/- 2.4 fmol mg-1 wet mass, corresponding to 94 sites per square micrometre for fibres whose mean diameter after fixation and embedding was estimated as 16 micron.     

Microdosimetry and chromosome aberrations: Effects of 230 keV neutrons on Vicia faba chromosomes

Physical energy deposition events have been related to sub-nuclear cytological events (chromosomal changes) in metaphases sequentially accumulated from the latter part of the cell cycle of Vicia faba. 230 keV neutrons produce about 0.4 recoil protons per late interphase nucleus per rad with the majority of protons travelling 1 to 2 microns from their origin, depositing energy at around 90 keV per micron. The frequency of induced aberrations is basically linear with dose, though varying through consecutive cell sampling periods because of differential induced mitotic delay. Distributions of chromosomal aberrations and total cytological events are overdispersed in relation to the Poisson distribuyion indicating that some proton recoils produce multiple events. When gaps and aberrations within chromosomes and multiple aberrations between chromosomes, are considered as discrete events, distributions follow Poisson expectations. About 40% of proton recoils result in observable cytological change. The highly energetic proton recoils (～90 keV per micron) which can induce multiple events are the ones most likely to produce effects which result in cell death. The sphere of influence of the proton recoils is probably adequately estimated from their range (～1 to 2 μm) since it seems compatible with the spatial proximity of the initial components of the resultant chromosome aberrations.