Nuclear Surface Area Research Articles

Confinement-sensitive volume regulation dynamics via high-speed nuclear morphological measurements

Diverse tissues in vivo present varying degrees of confinement, constriction, and compression to migrating cells in both homeostasis and disease. The nucleus in particular is subjected to external forces by the physical environment during confined migration. While many systems have been developed to induce nuclear deformation and analyze resultant functional changes, much remains unclear about dynamic volume regulation in confinement due to limitations in time resolution and difficulty imaging in PDMS-based microfluidic chips. Standard volumetric measurement relies on confocal microscopy, which suffers from high phototoxicity, slow speed, limited throughput, and artifacts in fast-moving cells. To address this, we developed a form of double fluorescence exclusion microscopy, designed to function at the interface of microchannel-based PDMS sidewalls, that can track cellular and nuclear volume dynamics during confined migration. By verifying the vertical symmetry of nuclei in confinement, we obtained computational estimates of nuclear surface area. We then tracked nuclear volume and surface area under physiological confinement at a time resolution exceeding 30 frames per minute. We find that during self-induced entrance into confinement, the cell rapidly expands its surface area until a threshold is reached, followed by a rapid decrease in nuclear volume. We next used osmotic shock as a tool to alter nuclear volume in confinement, and found that the nuclear response to hypo-osmotic shock in confinement does not follow classical scaling laws, suggesting that the limited expansion potential of the nuclear envelope might be a constraining factor in nuclear volume regulation in confining environments in vivo.

Rethinking nuclear shaping: insights from the nuclear drop model.

Changes in the nuclear shape caused by cellular shape changes are generally assumed to reflect an elastic deformation from a spherical nuclear shape. Recent evidence, however, suggests that the nuclear lamina, which forms the outer nuclear surface together with the nuclear envelope, possesses more area than that of a sphere of the same volume. This excess area manifests as folds/wrinkles in the nuclear surface in rounded cells and allows facile nuclear flattening during cell spreading without any changes in nuclear volume or surface area. When the lamina becomes smooth and taut, it is inextensible, and supports a surface tension. At this point, it is possible to mathematically calculate the limiting nuclear shape purely based on geometric considerations. In this paper, we provide a commentary on the "nuclear drop model" which seeks to integrate the above features. We outline its testable physical properties and explore its biological implications.

Machine learning-based morphological quantification of replicative senescence in human fibroblasts.

Although aging has been investigated extensively at the organismal and cellular level, the morphological changes that individual cells undergo along their replicative lifespan have not been precisely quantified. Here, we present the results of a readily accessible machine learning-based pipeline that uses standard fluorescence microscope and open access software to quantify the minute morphological changes that human fibroblasts undergo during their replicative lifespan in culture. Applying this pipeline in a widely used fibroblast cell line (IMR-90), we find that advanced replicative age robustly increases (+28-79%) cell surface area, perimeter, number and total length of pseudopodia, and nuclear surface area, while decreasing cell circularity, with phenotypic changes largely occurring as replicative senescence is reached. These senescence-related morphological changes are recapitulated, albeit to a variable extent, in primary dermal fibroblasts derived from human donors of different ancestry, age, and sex groups. By performing integrative analysis of single-cell morphology, our pipeline further classifies senescent-like cells and quantifies how their numbers increase with replicative senescence in IMR-90 cells and in dermal fibroblasts across all tested donors. These findings provide quantitative insights into replicative senescence, while demonstrating applicability of a readily accessible computational pipeline for high-throughput cell phenotyping in aging research.

Nuclear shapes are geometrically determined by the excess surface area of the nuclear lamina.

Introduction: Nuclei have characteristic shapes dependent on cell type, which are critical for proper cell function, and nuclei lose their distinct shapes in multiple diseases including cancer, laminopathies, and progeria. Nuclear shapes result from deformations of the sub-nuclear components-nuclear lamina and chromatin. How these structures respond to cytoskeletal forces to form the nuclear shape remains unresolved. Although the mechanisms regulating nuclear shape in human tissues are not fully understood, it is known that different nuclear shapes arise from cumulative nuclear deformations post-mitosis, ranging from the rounded morphologies that develop immediately after mitosis to the various nuclear shapes that roughly correspond to cell shape (e.g., elongated nuclei in elongated cells, flat nuclei in flat cells). Methods: We formulated a mathematical model to predict nuclear shapes of cells in various contexts under the geometric constraints of fixed cell volume, nuclear volume and lamina surface area. Nuclear shapes were predicted and compared to experiments for cells in various geometries, including isolated on a flat surface, on patterned rectangles and lines, within a monolayer, isolated in a well, or when the nucleus is impinging against a slender obstacle. Results and Discussion: The close agreement between predicted and experimental shapes demonstrates a simple geometric principle of nuclear shaping: the excess surface area of the nuclear lamina (relative to that of a sphere of the same volume) permits a wide range of highly deformed nuclear shapes under the constraints of constant surface area and constant volume. When the lamina is smooth (tensed), the nuclear shape can be predicted entirely from these geometric constraints alone for a given cell shape. This principle explains why flattened nuclear shapes in fully spread cells are insensitive to the magnitude of the cytoskeletal forces. Also, the surface tension in the nuclear lamina and nuclear pressure can be estimated from the predicted cell and nuclear shapes when the cell cortical tension is known, and the predictions are consistent with measured forces. These results show that excess surface area of the nuclear lamina is the key determinant of nuclear shapes. When the lamina is smooth (tensed), the nuclear shape can be determined purely by the geometric constraints of constant (but excess) nuclear surface area, nuclear volume, and cell volume, for a given cell adhesion footprint, independent of the magnitude of the cytoskeletal forces involved.

Intraductal Carcinoma of the Prostate: Extreme Nuclear Size Is Not a Diagnostic Parameter.

High-grade prostatic adenocarcinoma involving duct/acinar structures is labeled intraductal carcinoma of the prostate (IDCP). As numerous studies have shown that IDCP is associated with high stage disease with a significant negative impact on cancer-specific survival, accurate diagnosis is crucial to ensure appropriate patient management. The definition of IDCP recommended by 2016 World Health Organization (WHO) classification suggests that cases of IDCP with micropapillary or loose cribriform architecture without comedonecrosis should have cells with ≥6× nuclear enlargement. It is unclear how this size criterion was derived and which of the parameters of nuclear size (nuclear diameter, nuclear surface area, or nuclear perimeter) it relates to. To evaluate the extent of nuclear enlargement in IDCP, we performed morphometric analyses relating to each of these parameters in 100 radical prostatectomy specimens. One hundred nuclei from foci of IDCP and 50 nuclei from foci of normal luminal epithelium were examined for each patient. Diagnosis of IDCP was based on cells with definite features of carcinoma present within duct/acinar structures. Comparing the means of each of the parameters between IDCP cells and benign luminal cells, there was a statistically significant enlargement in nuclear perimeter (P<0.0005), nuclear area (P<0.0005), and nuclear diameter (P<0.0005); however, the difference in mean nuclear size was limited to factors of 1.3×, 1.6×, and 1.3×, respectively. Three patients each had rare large nuclei (largest perimeter 45, 45, and 44 μm; maximum nuclear area 135, 136, and 136 μm2; and the largest diameter 18 µm in each). For these rare cells, the nuclear size difference, when compared with benign nuclei was; nuclear perimeter 2.0×, 2.1×, and 2.1×; nuclear area 3.6×, 3.8×, and 3.8×; and nuclear maximum diameter 3.0×, 2.5×, and 2.5×. The definition of nuclear enlargement of ≥6× was not reached in any of our cases, all of which clearly showed features of duct invasive carcinoma. In these cases, reliance on nuclear size criteria would have resulted in underdiagnosis of IDCP. This is of concern as failure to recognize IDCP, particularly in needle biopsies, could lead to delays in the timely treatment of aggressive high-grade prostate cancer, resulting in cancer progression and suboptimal patient oncological outcomes.

Nuclear Deformation Lets Cells Gauge Their Physical Confinement.

How cells sense their physical microenvironment remains incompletely understood. In two recent Science articles, Lomakin etal. (2020) and Venturini etal. (2020) demonstrate that progressive nuclear deformation associated with cellular confinement triggers intracellular events that promote cell contractility and migration, revealing the nucleus to serve as a central mechanosensor.

Application of p16, p63, cyclin D1 immunostaining and nuclear morphometric analysis for assessment of cervical dysplasia

Background/Aim. Human papilloma virus (HPV) infection is the main etiological factor for the development of cervical precancerous dysplastic squamous intraepithelial lesions (SIL). The virus oncoproteins affect several proteins included in cell proliferation. The aim of this study was to evaluate application of immunohistochemical markers related to proteins of the cell cycle and, also, application of nuclear morphometric analysis for assessment of cervical dysplasia. Methods. Retrospective study included 78 women with detection of presence of high-risk HPV by polymerase chain reaction (PCR), with histopathology diagnosis low-grade SIL (LSIL) or high-grade SIL (HSIL). Immunohistochemical staining for p16, p63, cyclin D1 and morphometric analysis of the nuclear surface area were performed. The control group consisted of ten women without SIL and without HPV infection. This study was conducted in accordance with the Helsinki Declaration. Results. Comparing immunohistochemical expression of p16 and p63, highly statistically significant differences (p &lt; 0.001) were established among the control, LSIL and HSIL groups, while cyclin D1 showed significant statistical difference (p &lt; 0.05). Great variations were observed in nuclear morphology and nuclear surface area that had highly statistically significant differences (p &lt; 0.001) among the control, LSIL and HSIL groups. Conclusion. This study demonstrated that immunohistochemical analysis of p16, p63 and cyclin D1 are important for diagnosis of dysplastic changes in cervical epithelium. Also, morphometric analysis of the nuclear surface area demonstrated a high significance for diagnosis of cervical dysplasia.

Metabolic enzyme profile, behavioural changes and morphophysiological parameters of African catfish Clarias gariepinus juveniles in response to burnt waste tyres

Water-soluble fractions (WSFs) of waste burnt tyres (WBTs) were tested on enzymatic activities, behavioural changes and histology of gills and liver of African catfish Clarias gariepinus juveniles for 96 h at 5.00, 10.00, 15.00, 20.00 and 25.00 g/L in a non renewable static bioassay. Water quality parameters of the experimental media showed significant (p < 0.05) decrease in pH and dissolved oxygen content and increased carbon dioxide and alkalinity levels compared to the control. The 96-h LC50 of WSFs of WBTs to C. gariepinus was calculated as 11.22 g/L. There were remarkable abnormal behaviours exhibited by the exposed fish as well as alteration in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase activities in gills and liver in a dose-dependent manner. The exposure also led to changes in primary and secondary gill lamellar hypertrophy and hyperplasia, partial fusion of apical lamellar, cytoplasmic and nuclear degeneration of parenchyma cells, as well as cytoplasmic vacoulation, sinusoidal oedema and nuclear and periportal hepatocyte degeneration of the liver. Hepatomorphometry of the fish revealed significant (p < 0.05) increase in nuclear surface area as well as nuclear diameter of the hepatic cells. BWTs could be deleterious to aquatic biota including fish and could be adopted as biomarkers for assessing the quality of aquatic ecosystem.

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics.

Nuclei are often under external stress, be it during migration through tight constrictions or compressive pressure by the actin cap, and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area, respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

Nanocarrier anticancer drug-conjugates cause higher cellular deformations: culpable for mischief

Here we report nanocarrier-anticancer drug conjugates culpable for cellular deformations, critically evidenced through image-based analysis as a measure of karyoplasmic ratio (KR) and nuclear surface area (NSA). Multiwalled carbon nanotubes (MWCNTs) were coordinated additionally with Fe3O4 nanoparticles (NPs) to evaluate the symbiotic influence, and further conjugated to Dox for evaluating the cellular kinetics and for measuring cell deformations. Cellular entry kinetics of the CNT (CNT-Dox and CNT-Cys-Fe3O4-Dox) nanocarriers and their efficiency in nuclear localization were evaluated using cervical cancer (HeLa) cells. Of note, the Dox-bound nanocarriers showed significantly enhanced cell toxicity over the free form of the drug. CNT-Dox and CNT-Cys-Fe3O4-Dox influx occurred within 4 hours, while maximum cellular retention of Dox was observed for CNT-Dox at 24 h. However, the highest KR (∼0.51) was observed for CNT-Dox within 8 hours indicating similar cellular deformations using nanocarrier anticancer drug-conjugates to that of free Dox (KR ∼0.50) at 4 hours. In addition, we observed increased NSA at 4 h in Dox treatment whereas in the case of the Dox conjugated nanocarrier, increased NSA was noted at 8 h treatment. At 8 h exposure of HeLa cells with Dox conjugates, we observed that the cells fall into distinct regions of the morphospace with respect to KR and NSA. Conclusively, nano delivery systems considered for clinical and biomedical translations must take into account the possible negative influences imparting higher cellular deformations and secondary adverse effects over the free form of the drug.

A homeostatic mechanism rapidly corrects aberrant nucleocytoplasmic ratios maintaining nuclear size in fission yeast.

ABSTRACTNuclear size scales with cell size across a wide range of cell types. The mechanism by which this scaling is maintained in growing cells remains unclear. Here, we investigate the mechanism of nuclear size homeostasis in the simple eukaryote fission yeast, by monitoring the recovery of aberrant nuclear volume to cell volume (N/C) ratios following perturbation. We demonstrate that both high and low N/C ratios correct rapidly, maintaining nuclear size homeostasis. We assess the kinetics of nuclear and cellular growth and of N/C ratio correction, and demonstrate that nuclear and cellular growth rates are not directly coupled. We propose that the mechanism underlying nuclear size homeostasis involves multiple limiting factors implicated in processes including nucleocytoplasmic transport, lipid biogenesis and RNA processing. We speculate that these link cellular size increases to changes in nuclear contents, which in turn lead to changes in nuclear membrane surface area. Our study reveals that there is rapid nuclear size homeostasis in cells, informing understanding of nuclear size control and size homeostasis of other membrane-bound organelles.

Nuclear Migration: An Indicator of Plant Salinity Tolerance in vitro.

In order to understand the mechanisms underlying acquisition of tolerance to salinity, we recently produced callus tissues of tobacco and Medicago truncatula resistant to NaCl-induced salt stress following application of a step-up recurrent selection method. The effects of salinity on cell size are known, but those on cell morphometry including cell and nuclear surface area and position of nuclei within salt stress resistant cells were never studied before. This work fills that gap, using suspension cultured cells of M. truncatula A17 initiated from callus, and Nicotiana tabacum BY-2 cell line resistant to increasing NaCl concentrations up to 150 mM NaCl. The surface area of salinity resistant cells of M. truncatula A17 and N. tabacum BY2 and their nuclei, produced by step-up recurrent selection, were reduced, and cells elongated as NaCl increased, but these parameters proved to be unreliable in explaining cell survival and growth at high NaCl. Conversely, nuclei of resistant cells migrated from the center to the periphery of the cytoplasm close to the walls. Nuclear marginalization was for the first time observed as a result of salt stress in plant cells, and could be a novel helpful morphological marker of acquisition of salinity tolerance.

Immunohistochemical and electron microscopic morphometric image analysis of hepatocellular carcinoma in association of HCV infection

ABSTRACTEarly detection of hepatocellular carcinoma (HCC) is crucial for successful therapy. The present work examined the value of ultrastructural morphometric image analysis of hepatocyte nuclei in patients with chronic hepatitis C virus (HCV) versus HCC cases with chronic HCV and the corresponding surgical tumor-free safe margins (TFMs), to highlight any early predictive signs of neoplastic cellular transformation. This work also performed an immunohistochemical assessment of cytokeratin 19 (CK19) and Ki-67-positive cells to visualize any associated proliferative activity in the examined groups.The results showed significant decrease in the hepatocyte nuclear surface areas in the HCC and TFMs versus those in the HCV cases. The hepatocyte nucleolar surface area was significantly increased in the HCC cases versus that in the HCV cases. This increase was associated with a significant increase in Ki-67-positive cells in the HCC cases compared to those in the other groups. Conversely, the mean number of CK 19-positive cells was significantly reduced in the HCC cases compared to the cell numbers in TFMs and HCV cases with severe hepatic fibrosis. Liver progenitor cells (LPCs) were discerned in the reactive ductules and canaliculo-ductular junctions that characterized TFMs. LPCs were sporadically distributed in the liver lobules and reactive bile ductules in the HCC samples.In conclusion, CK 19 represents an important marker for distinguishing between dysplastic and malignant liver nodules. Electron microscopic morphometric image analysis may be considered as adjunct factor for assessing hepatocyte malignant transformation. Wider scale studies are needed to authenticate these results.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Effects of hydrochloric acid treatment on structure characteristics and C2H4 adsorption capacities of Ünye bentonite from Turkey: a combined FT-IR, XRD, XRF, TG/DTA and MAS NMR study

In this study, a bentonite sample from Unye, Turkey was treated with various HCl solutions (0.1, 1.0 and 5.0 M) at 90 °C during 3 and 6 h. X-ray diffraction (XRD), X-ray fluorescence (XRF), differential thermal analysis (DTA), thermogravimetric analysis (TG), Fourier transform infrared (FT-IR), magic angle spinning nuclear magnetic resonance (29Si and 27Al MAS NMR) and surface area measurement methods were employed in order to investigate the structural and thermal changes occurring as a result of the acid activation. The data for the adsorption of C2H4 obtained at 273 K and pressures up to 100 kPa correlated with structural properties of the acid treated bentonite samples. With the increase of both concentration of acid solution and treatment time, the specific surface area values and the retentions of C2H4 gas of bentonite samples increased. Bentonite treated with 5.0 M HCl for 6 h adsorbed C2H4 most effectively.

Measurement of In Vivo Three-Dimensional Corneal Cell Density and Size Using Two-Photon Imaging in C57BL/6 Mice

ABSTRACTPurpose: To measure the cell size and cell density in five layers of the central cornea in the widely used inbred C57BL/6 mouse strain using in vivo three-dimensional (3D) two-photon (2PH) imaging.Methods: Corneas were scanned using a 2PH laser scanning fluorescence microscope after staining with plasma membrane stain and Hoechst 33342. Good quality 3D images were selected for the cell density and cell size analysis. Cell density was determined by counting the cell nuclei in a predefined cube of 3D images. Cell size measurements, including cell surface area, cell volume, nuclear surface area and nuclear volume, were automatically quantified using the Imaris software. The cell and nuclear surface-area-to-volume ratio (S:V ratio) and the cell nuclear-cytoplasmic ratio (N:C ratio) were calculated.Results: The highest cell density was observed in the basal epithelium and the lowest in the posterior stroma. The highest cell surface area was found in the anterior stroma, and the highest cell volume was observed in the superficial epithelium. The lowest cell surface area and cell volume were both found in the basal epithelium. The highest S:V ratio was observed in the basal epithelium and the lowest in the superficial epithelium. The highest cell nuclear surface area and volume were both observed in the superficial epithelium and the lowest in the basal epithelium. The highest cell nuclear S:V ratio was observed in the basal epithelium and the lowest in the superficial epithelium. The highest N:C ratio was found in the basal epithelial cells and the lowest in the posterior keratocytes.Conclusions: We are the first to quantify the cell density and size parameters, including cell surface area and volume, cell nuclear surface area and volume, and the S:V ratio, in the five layers of the central cornea. These data provide important cell morphology features for the study of corneal physiology, pathology and disease in mice, particularly in C57BL/6 mice.

In vivo and three-dimensional measurement of corneal cell density and size in BALB/c mice

Background Corneal cell size and density are among the primary parameters that determine the structural integrity and normal physiological function of the cornea.Recently, BALB/c mice have become the most widely used laboratory animal in corneal researches and studies.An understanding of normal corneal cell size, morphology and cell density is desirable for future comparisons when mouse models are used in corneal research. Objective The aim of this study was to measure the cell size and cell density in three layers of the central cornea in the widely used inbred BALB/c mice strain based on in vivo three-dimensional (3D) two-photon (2PH) imaging. Methods Corneal endothelium layer, corneal stromal layer and corneal epithelium layer were sequentially scanned in six BALB/c mice by using a 2PH laser scanning microscope after staining with plasma membrane stain and Hoechst 33342 by intrastromal injection with the scanning thickness 2 μm for each layer, pixels 512×512 and 12 bit.Good quality 3D images were selected for the cell density and cell size analyses.Cell density was determined by counting the cell nuclei in a predefined cube of 3D images.Cell size measurements including cell surface area, cell volume, nuclear surface area, and nuclear volume were automatically quantified using the Imaris software.The cell and nuclear surface-area-to-volume ratio and the cell nuclear-to-cytoplasmic ratio were calculated. Results Corneal basal epithelium layer and endothelium layer appeared the monolayer cells, while middle stromal layer showed the double-layer cells under the 2PH laser scanning microscope.The cell density was (108.00±18.97)×104/mm3 in the basal epithelium layer, which was significantly higher than (9.27±0.48)×104/mm3 in the middle stromal layer and (22.30± 2.28) ×104/mm3 in the endothelium layer (F=141.592, P=0.000). The cell surface area and volume were highest in the middle stromal layer and lowest in the basal epithelium layer, with significant differences among the three layers (F=2 222.000, 598.504, both at P=0.000). The area/volume ratios of cells were (0.80±0.13), (0.51±0.07) and (0.40±0.04)/μm in the basal epithelium layer, middle stromal layer and endothelium layer, with the highest ratio in the basal epithelium layer and lowest ratio in the endothelium layer (F=127.075, P=0.000). The cell nuclear surface area and volume were highest in the endothelium and lowest in the basal epithelium.The cell nuclear surface area/volume ratio was highest in the basal epithelial cell and lowest in the endothelial cell.In addition, the nucleus/cytoplasm ratio was highest in the endothelial cells and lowest in the middle keratocytes. Conclusions 2PH laser scanning microscope can quantitatively measure the parameters of corneal cells in vivo in BALB/c mice, including cell surface area and volume, cell nuclear surface area and volume.These data provide important cell morphology features for the study of corneal physiology, pathology and disease in mice, particularly in BALB/c mice. Key words: Cornea/anatomy & histology; Cell count; Cell size; Mice, inbred BALB/c; Microscopy, fluorescence, two-photon

강도다리(Platichthys stellatus)에 대한 세포유전학적 연구

【Cytogenetic analysis was conducted to obtain basic information for chromosome manipulation of starry flounder Platichthys stellatus. Nuclear surface area and volume of erythrocyte were $7.60{\pm}0.93{\mu}m^2$ and $12.80{\pm}1.75{\mu}m^3$ , respectively. The haploid DNA content of the species was 0.66 pg/haploid cell which correspond to 93% of olive flounder Paralichthys olivaceus. A karyotype analysis was also carried out with the species using conventional staining and Ag-NOR banding techniques. It was consisted of 48 acrocentric chromosomes and inter-sex or intra-individual polymorphism was not detected in all specimens analyzed. The NOR regions, appearing a terminal position of the short arm of the smallest acrocentric pairs.】

Fluoride induced alterations in erythrocyte and related parameters of an Air-breathing fish, Channa punctatus (Bloch)

The effect of various concentrations of fluoride (NaF) (10, 20, 30 and 40ppm) after 15 days of exposure on some haematobiochemical parameters of Channa punctatus (Bloch) has been studied. Changes in the shape and size, cell membrane and nucleus of the R.B.C. were observed. Clumping of the R.B.C. cells was noted. Clumping becomes prominent in higher concentrations. The TEC, Hb, PCV, MCV, MCH and MCHC progressively decreased with an increase in the concentration of fluoride. The length/ breadth ratio of erythrocyte and erythrocyte nucleus do not depart significantly from normal values and as such there is no significant change in erythrocyte and nuclear surface area.

Effect of morphactin (chlorfluorenol IT 3456) on the mitotic activity and cell growth in roots of Pisum sativum L.

Morphactin in a concentration of 100 ppm does not retard pea root growth, however, it reduced the frequency of cell division and shifted the main wave of mitoses in the 24 h period from 1 to 2 mm of root segment. The mean cell cycle duration is prolonged from 14 h in the control to 22 h in morphactin-treated roots. In the presence of morphactin the decrease of &lt;sup&gt;3&lt;/sup&gt;H-thymidine incorporation and diminution of labelling index is accompanied by reduction of the nuclear surface area. The described changes are not accompanied by shortening of cell length. The results obtained suggest that morphactin disturbs the mechanisms regulating the initiation of S phase and its regular course. Moreover, it inhibits the endomitotic replication of DNA.