Average Nuclear Volume Research Articles

CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Spatial relationship between chromosomal domains in diploid and autotetraploid Arabidopsis thaliana nuclei

ABSTRACTPolyploids constitute more than 80% of angiosperm plant species. Their DNA content is often further increased by endoreplication, which occurs as a part of cell differentiation. Here, we explore the relationship between 3D chromatin architecture, number of genome copies and their origin in the model plant, Arabidopsis thaliana. Spatial proximity between pericentromeric, interstitial and subtelomeric domains of chromosomes 1 and 4 was quantified over a range of distances. The results indicate that average nuclear volume as well as chromatin density increase with the genome copy number. Similar dependence is observed when association of homologous chromosomes (in 2C/ endopolyploid nuclei) and sister chromatid separation (in endopolyploid nuclei) is studied. Moreover, clusters of chromosomal domains are detectable at the spatial scale above microscopy resolution. Subtelomeric, interstitial and pericentromeric chromosomal domains are affected to different extent by these processes, which are modulated by endopolyploidy. This factor influences fusion of heterochromatin as well. Nonetheless, local chromatin architecture of Arabidopsis thaliana depends mainly on endopolyploidy level, and to lesser extend on polyploidy.

92 NUCLEAR INVAGINATIONS ADAPT TO RABBIT EARLY EMBRYONIC DEVELOPMENT

Nuclear invaginations carrying nuclear pores may facilitate increased mRNA export and protein import to areas inside the nucleus remote from the nuclear border. In this study on rabbit embryos, we investigated whether large early embryonic nuclei and the increased import/export demands around major embryonic genome activation (MGA) at the 8-cell stage affected the quantity of nuclear invaginations carrying nuclear pores. To achieve this objective, we used super-resolution 3-dimensional structured illumination microscopy on 10 pronuclei or nuclei per stage of 23 in vivo-fertilized and in vitro-cultured embryos stained with antibodies for the nucleoporin NUP153 and lamin B and stained with 4′,6-diamidino-2-phenylindole (DAPI) for chromatin. Statistical comparisons between stages were performed using the Wilcoxon rank-sum test. At the zygote stage, the female pronucleus displayed on average 16.5 and the male pronucleus featured on average 15.25 wide and narrow nuclear envelope invaginations, carrying large or tiny amounts of cytoplasm. Subsequent stages showed predominantly wide invaginations targeting nucleoli. The contact areas between nucleoli and invaginations were free of nuclear pores. In contrast, narrow invaginations, which are the almost exclusive type of invaginations during cattle and mouse pre-implantation development, were not in contact with nucleoli. At the 2-cell stage, the number of invaginations increased to an average of 27.3 invaginations per nucleus (P < 0.05) and increased again to peak at the 4-cell stage with 51.2 invaginations per nucleus (P < 0.01). At the 8-cell stage (MGA), the amount of nuclear invaginations was reduced to 25.4 invaginations per nucleus (P < 0.01). The reduced number of nuclear invaginations at the 8-cell stage could be associated with a significant decrease in average nuclear volume from 2593 µm3 at the 4-cell stage to 960 µm3 at the 8-cell stage (P < 0.001) and a subsequent reduced average distance from areas inside the nucleus to the nuclear border. Nuclear invagination numbers continued their decline at the 21-cell stage with 5.2 invaginations per nucleus (P < 0.001), whereas nuclear volumes decreased to 618 µm3 (P < 0.001). The morula stage, with 6.9 invaginations per nucleus (P = 0.9), and the blastocyst stage, with 4.5 invaginations per nucleus (P = 0.5), showed no more significant changes. Large NUP153 cytoplasmic clusters present before MGA may represent a maternally provided NUP153 deposit. MGA may mark the switch from the use of a NUP153 deposit to on-demand production. Additionally, over- and under-representation analyses on mitotic blastomeres revealed that NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina (P < 0.001; chi-squared goodness-of-fit test). In conclusion, rabbit embryonic development is accompanied by stage-dependent changes of the amount of nuclear invaginations carrying nuclear pores. Although cattle and mouse embryos almost exclusively feature narrow invaginations restricted to the nuclear periphery and not in contact with nucleoli, rabbit embryos feature additional wide invaginations that can reach across the nucleus and target nucleoli.

MP41-19 THREE-DIMENSIONAL MOLECULAR CHARACTERIZATION AND ISOLATION OF CIRCULATING TUMOR CELLS IN METASTATIC PROSTATE CANCER PATIENTS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2014MP41-19 THREE-DIMENSIONAL MOLECULAR CHARACTERIZATION AND ISOLATION OF CIRCULATING TUMOR CELLS IN METASTATIC PROSTATE CANCER PATIENTS Tadeusz Kroczak, Julius Adebayo Awe, Adam Yan, Nidhi Shah, Alex Kuzyk, Jeff Saranchuk, Sabine Mai, and Darrel Drachenberg Tadeusz KroczakTadeusz Kroczak More articles by this author , Julius Adebayo AweJulius Adebayo Awe More articles by this author , Adam YanAdam Yan More articles by this author , Nidhi ShahNidhi Shah More articles by this author , Alex KuzykAlex Kuzyk More articles by this author , Jeff SaranchukJeff Saranchuk More articles by this author , Sabine MaiSabine Mai More articles by this author , and Darrel DrachenbergDarrel Drachenberg More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1236AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Circulating tumor cells (CTCs) are emerging as a promising bio-marker in prostate cancer screening and in monitoring of disease progression. In order to utilize CTCs clinically, an efficient and reliable method of CTC isolation must be developed. Upon isolation of CTCs, analysis of the three-dimensional (3D) nuclear organization of telomeres can be used to further profile and stratify prostate cancer patients. The relative aggressiveness of a tumor can be correlated to the degree of chromosomal instability seen within a given cell. We show that metastatic prostate cancer patients display CTCs and telomeric profiles that correlate to high-risk prostate cancer phenotypes. Methods CTCs from ten consecutive patients presenting with metastatic prostate cancer were isolated using the size based ScreenCell filtration technique. Cytokeration 8,18, 19 immunostaining and 3D quantative fluorescence in situ hybridization was performed on the isolated CTCs followed by 3D image acquisition using a Carl Zeiss AxioImager Z2 microscope. Quantitative image analysis with TeloviewTM and Teloscan were then preformed to obtain 3D telomere profiles and to identify the number of CTCs. Results Our data shows that CTCs are present in abundance and can be isolated in patients with metastatic prostate cancer. Furthermore, these CTCs have similar telomere profiles when comparing the following statistical parameters: percentage of cells with aggregates, average number of telomeres per cell, average number of aggregates per cell and average nuclear volume. Conclusions This proof of principle study shows for the first time that CTCs in metastatic prostate cancer patients can be reliably isolated and characterized by 3D nuclear telomere profiling using ScreenCell filters as well as presenting similar telomeric profiles. These findings show that prostate cancer CTCs and telomeric analysis have the potential to become a biomarker for tumor stage and progression. Our results could be used as positive controls when looking at less aggressive prostate cancer phenotypes. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e458 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Tadeusz Kroczak More articles by this author Julius Adebayo Awe More articles by this author Adam Yan More articles by this author Nidhi Shah More articles by this author Alex Kuzyk More articles by this author Jeff Saranchuk More articles by this author Sabine Mai More articles by this author Darrel Drachenberg More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The size of the nucleus increases as yeast cells grow.

It is not known how the volume of the cell nucleus is set, nor how the ratio of nuclear volume to cell volume (N/C) is determined. Here, we have measured the size of the nucleus in growing cells of the budding yeast Saccharomyces cerevisiae. Analysis of mutant yeast strains spanning a range of cell sizes revealed that the ratio of average nuclear volume to average cell volume was quite consistent, with nuclear volume being approximately 7% that of cell volume. At the single cell level, nuclear and cell size were strongly correlated in growing wild-type cells, as determined by three different microscopic approaches. Even in G1-phase, nuclear volume grew, although it did not grow quite as fast as overall cell volume. DNA content did not appear to have any immediate, direct influence on nuclear size, in that nuclear size did not increase sharply during S-phase. The maintenance of nuclear size did not require continuous growth or ribosome biogenesis, as starvation and rapamycin treatment had little immediate impact on nuclear size. Blocking the nuclear export of new ribosomal subunits, among other proteins and RNAs, with leptomycin B also had no obvious effect on nuclear size. Nuclear expansion must now be factored into conceptual and mathematical models of budding yeast growth and division. These results raise questions as to the unknown force(s) that expand the nucleus as yeast cells grow.

95 OBSERVATION OF CHROMOSOME DECONDENSATION, HISTONE H3 MODIFICATION, AND HP1 PROTEIN IN MOUSE CLONED EMBRYOS FOLLOWING INHIBITION OF HISTONE DEACETYLATIONS AND CYCLIN-DEPENDENT KINASE

Low chromosome decondensation and hypoacetylation and hypermethylation of histones characterize the incomplete reprogramming of cloned embryos during the first mitotic prophase. Our study looks at the effect of histone deacetylase inhibitors (HDACi), Trichostatin A (TSA), APHA Compound 8 (APHA), and cyclin-dependent kinase inhibitor (CDKi) roscovitin (ROS), on nuclear reprogramming in cloned mouse embryos during the first mitotic prophase. Oocytes were collected from female B6D2F1 mouse cumulus cells collected from female B6D2F1 and ICR, and fibroblasts from male GFP-ICR. We performed somatic cell nuclear transfer (SCNT) using a piezo-actuated micromanipulator system; the SCNT oocytes were subsequently activated for 6 h with 5 mM SrCl2 in Ca2+-free CZB medium (CZB) supplemented individually with 100 nM TSA, 250 nM APHA, or 100 nM ROS or in combination with TSA+ROS or APHA+ROS. Activated SCNT embryos were then cultured in KSOM medium with the same concentrations of TSA, APHA, ROS, TSA+ROS, or APHA+ROS for 2 h. Following treatment, we cultured the cloned embryos in KSOM for in vitro development. In the first experiment, the levels of chromosome decondensation during the first mitotic prophase in these cloned embryos were performed, based on the ratio of nuclear volume, at 6 h and 10 h after activation. We calculated the average nuclear volume � SD for each treatment, and these were subsequently compared to control values (100%, without treatment). We next turned to examining the intensity of methyl H3-K9, acetyl H3-K9, and HP1β of cloned embryos at 6 h and 10 h after activation by immunostaining. Images of immunostained embryo nuclei were acquired using an OLYMPUS Fluoview FV 1000 confocal system. The distribution of fluorescence intensities from 5 different regions of the nucleus were determined using Fluoview FV 1.4a software. Student's t-test was used to calculate the significance of differences between groups in the experiment. We repeated each experiment 4 times to obtain 40 nuclear transfer embryos per treatment. The present results show that global histone hyperacetylation and hypomethylation can be characterized by an increase in H3-K9 acetylation and a decrease in H3-K9 methylation in the presence of HDAC inhibitors. The levels of all isoforms of HP1β, however, were not reduced following inhibition of HDAC. This was in contrast to the high decondensation observed in interphase chromatin, leading to a significant increase of pronuclear volumes and a decrease in H3-K9 methylation in cloned embryos in the presence of ROS, although we were unable to obtain hyperacetylation and HP1β did not change. From these results, we conclude that the combined inhibition of histone deacetylases and cyclin-dependent kinase for 8 h following NT caused an increase in somatic chromosome decondensation, hyperacetylation, and demethylation of histone H3-K9 during the first mitotic cell cycle in cloned mouse embryos. Our findings strongly suggest that there is no correlation of HP1β involved in hyperacetylation and demethylation of H3-K9.

Growth and morphological changes in the digestive tract of rainbow trout ( Oncorhynchus mykiss) and pacu ( Piaractus mesopotamicus) due to casein replacement with soybean proteins

The effect of diets in which 50% of casein-gelatin protein was replaced with extracted soybean meal (SBM) or soybean protein concentrate (SPC) on first-feeding rainbow trout and juvenile South American pacu was examined following 3–6 fold body weight gain. A casein-gelatin-based diet supplemented with essential amino acids, lipids and other ingredients was used as control. After 4-weeks feeding, rainbow trout growth was significantly depressed in both SBM- and SPC-replacement treatments whereas pacu, the adults of which are considered omnivorous or frugivorous, showed significantly improved weight gain on the SBM-replacement diet. The enterocytes of posterior intestine of all control fish, and SBM-fed pacu showed regular shapes. Their supranuclear regions contained numerous small absorptive vacuoles. In trout fed SPC and SBM diets, and in SPC-fed pacu, posterior intestine enterocytes were excessively vacuolized. The highest pancreas activity (measured as the number of proenzyme granules) occurred in control fish. The liver cells showed regular development in both species fed the control diet and in pacu fed SBM and SPC diets. On the contrary, the hepatocytes of SBM and SPC-based diet fed rainbow trout showed anomalies. In both species, the average hepatocyte nuclear volumes significantly differed among the feeding groups. The results of histological analyses indicated that absorption and transport of nutrients to liver and pancreas were affected by the presence of soybean products in experimental diets. The SBM diet was beneficial for pacu but adversely affected rainbow trout, while the SPC diet resulted in extensive pathologies of digestive tract and most likely affected nutrient utilization in both species.

Treatment of pregnant females with dexamethasone influences postnatal development of the adrenal medulla

In the light of the mutual dependence between the adrenal cortex and medulla, the aim of this work was to examine whether glucocorticoid treatment of pregnant rats affects the development of the adrenal medulla of their offspring in the postnatal period. Pregnant rats were treated with dexamethasone (Dx) in a daily dose of 0.3 mg Dx/kg b.w. during days 16-20 of gestation. The structure and function of the adrenal medulla of their 14-day-old offspring were estimated on the basis of the morphometric parameters of the gland, chromaffin cell mitotic index and adrenal gland adrenaline content. Stereological analysis was carried out at the light microscopic level, the mitotic index was determined by counting the number of metaphase arrested chromaffin cells following the administration of vincristine-sulphate, whereas adrenaline content in the adrenal gland was measured fluorimetrically. Plasma ACTH concentrations of the offspring were also determined by RIA. Long term Dx treatment of pregnant rats caused a significant decrease of the total volume of adrenal chromaffin tissue in the 14-day-old offspring as well as a reduction in the number of chromaffin cells and the average cell and nuclear volumes. The proliferative activity of the chromaffin cells was also lower than in the control offspring. These changes were accompanied by a significantly reduced adrenaline content in the adrenals. The results of this work show that glucocorticoid excess during the period of pregnancy when the fetal adrenal medulla is formed has a strong inhibitory effect on the adrenal medulla of the offspring at the age of 14 days.

Postnatal development of ovine seminiferous tubules: an electron microscopical and morphometric study

Corresponding to the increasing testicular volume and the histological appearance of the testicular parenchyma, the postnatal ontogenesis of the ovine testis can be divided into five phases. During the prebubertal period (phases 1-III), seminiferous tubules are solid and contain supporting (pre-Sertoli) cells as well as up to three types of germ cells: prespermatogonia I, II and spermatogonia precursor cells. In phase I, only prespermatogonia I are present and can usually be observed at the center of the seminiferous tubules. During phase II, prespermatogonia I migrate towards the basal lamina, divide and become prespermatogonia II. Those prespermatogonia I which are not successful in establishing contact with the tubular basal lamina degenerate. In phase III, prespermatogonia II divide and differentiate into cells which function as stem cells for spermatogenesis. Morphometric data corroborate the assumption of two types of prespermatogonia in the postnatal prepubertal ovine testis. Prespermatogonia I have nuclear volumes of about 480 microns 3 and cellular volumes of about 1200 microns3. In prespermatogonia II both volumes increase to about 920 microns3 and 1800 microns3 respectively. Adult A-spermatogonia are significantly smaller and possess an average nuclear volume of about 340 microns3 and an average cellular volume of about 800 microns3. Concomitanty with the formation of the tubular lumen in puberty (phase IV), supporting cells differentiate morphologically into typical Sertoli cells. Developmental events in the germ cell population are not yet synchronized. Adulthood (phase V) is characterized by complete spermatogenesis with all stages of the seminiferous epithelial cycle.

Measurement characteristics of stereological and histochemical methods in the diagnosis of thyroid tumours.

Seventy-two female mice of BALB/c strain were divided into three control and three perchlorate groups. The perchlorate groups drank 1.2% sodium perchlorate solution. After 8 or 32 weeks of the 46-week experiment one control and one perchlorate group were totally irradiated with 0.8 Gy on 5 consecutive days. Ten normal thyroid glands, 10 with hypertrophy and hyperplasia, 8 follicular adenomas and 10 follicular carcinomas were histologically and stereologically analysed. The results showed significant differences in the average nuclear volume (p < 0.05) and in the average number of the silver-stained nucleolar organiser regions (AgNORs) per nucleus (p < 0.005) between the follicular adenomas and the follicular carcinomas. The method of counting Ag-NORs per nucleus was more valid, more accurate and more economical than the method of measuring the average nuclear volume.

Quantitative and ultrastructural alterations in the lamina propria and Sertoli cells in human cryptorchid testes

A quantitative and ultrastructural study was performed on biopsies of human cryptorchid testes to investigate lesions in the lamina propria and Sertoli cells. Prepubertal cryptorchid testes (1-9 years of age) were classified into four groups: Type 1, testes with minimal lesions; Type II, testes with a moderate decrease in tubule diameter and spermatogonal number; Type III, testes with Sertoli cell hypoplasia and a marked reduction in tubule diameter and spermatogonal number; and Type IV, testes with Sertoli cell hyperplasia and a variable reduction in spermatogonal number. An increase in thickness of the lamina propria was found in Type II and III testes from 5 years of age onwards. These testes also showed a decrease in both the average number of peritubular cells per cross-sectioned tubule and in the average nuclear volume of these cells. Most of the postpubertal cryptorchid testes from 13- to 18-year-old youths presented a prepubertal pattern suggestive of delayed testicular maturation. Postpubertal testes from 19- to 27-year-old men were classified into three types: Type A testes showed complete spermatogenesis, mature Sertoli cells and no lesions in the lamina propria; Type B testes showed isolated spermatogonia, mature Sertoli cells, and a marked thickening of the lamina propria; and Type C testes showed isolated spermatogonia, hyperplasia of immature Sertoli cells, and a slightly thickened lamina propria. Maturation of the lamina propria was always associated with maturation of the Sertoli cells. Thickening of the lamina propria was associated with peritubular cell alterations consisting of decreases in the nuclear volume (average and total per testis) of peritubular cells and increases in the number of these cells per cross-sectioned tubule. The three types of adult cryptorchid testes appear to be the postpubertal transformation of Type 1 testes (Type A), Type II and Type III testes (Type B), and Type IV testes (Type C).

Rabbit endocervical epithelium: morphometric analysis of secretory cell populations.

In this report we quantitated ultrastructural changes in two cytologically distinct secretory cell populations from the rabbit endocervix. Type I and type II cells from estrous animals differ only in the presence of one or more empty cytoplasmic vacuoles in type II cells. Comparing type II cells from 5-day pseudopregnant (PSP) rabbits with type II cells from estrous controls, there is no increase (P greater than .05) in the average vacuole volume. When type I and type II cells from PSP animals are compared to cells from estrous controls, there is a decrease (P less than .01) in the average cell volume, a decrease (P less than .01) in the average nuclear volume, and a decrease (P less than .01) in the average granule volume. This reduction in the granule content of secretory endocervical cells was correlated with a dramatic decrease in protein glycosylation into the microsomal fraction. Serum estradiol concentrations for estrous (13.7 +/- 1.0 pg/ml) and PSP (18.1 +/- 1.5 pg/ml) animals were comparable. However, the 36-fold increase in serum progesterone concentrations for PSP (12.04 +/- 1.7 ng/ml) animals compared to estrous (0.33 +/- 0.1 ng/ml) animals may be responsible for the decrease in protein glycosylation.

Dipyridamole-induced capillary endothelial cell proliferation in the rat heart--a morphometric investigation.

20 male Wistar rats were randomly divided into two equal groups. Each experimental rat was injected with 4 mg . kg-1 dipyridamole ip twice daily for 4 weeks, the control rats received sham injections of an equal volume of saline. The rats were fixed by retrograde vascular perfusion. Left ventricular papillary muscles were sampled for light and electron microscopic morphometry. By means of a combination of serial section reconstruction and morphometry we could demonstrate that the number of capillary endothelial cell nuclei per tissue volume had increased by 37% (P less than 0.01), whereas the average nuclear volume remained constant. All other morphometric parameters (volume densities of myocardial cells, capillaries, cellular and non-cellular interstitial tissue, and myocardial cell nuclei; surface area, diameter, and density of capillaries; volume densities of myofibrils, mitochondria, and sarcoplasmic matrix; surface areas of the inner and outer mitochondrial membranes; dimensions of average mitochondrial section profiles) were unchanged. We conclude that chronic dipyridamole application induces proliferation of capillary endothelial cells in the rat myocardium, possibly by an increase of myocardial blood flow.

Karyometric studies of the effect of lead and cadmium in relation to the oxygen supply in the chloragocytes of tubifex tubifex müll

The average nuclear volume of the chloragocytes of T. tubifex was approximately doubled on the fourth to sixth days of environmental hypoxia, compared with that of chloragocytes living under properly aerated conditions. The nuclear expansion is probably closely related to the stimulation of haemoglobin synthesis by the low oxygen tension. Under hypoxic conditions 10 ppm Cd killed all worms, while under aerated conditions Cd evoked a similar degree of nuclear expansion to that evoked by hypoxia alone. Ten parts per million of lead had a lower stimulating effect than cadmium on the chloragocyte nuclei of aerated worms. However, it retarded the stimulation which occurred under hypoxic conditions. Results are discussed considering both the known metabolic pathways of T. tubifex and the effects of cadmium and lead in other animals.

Lymphocyte blastogenesis studied by volume spectroscopy.

The blastogenesis of human blood lymphocytes has been studied by measuring the distribution of cellular and nuclear volumes in concanavalin A (Con A)-stimulated leucocyte cultures by means of a modified Coulter counter. Agglutinated and adherent cells were dispersed by 3 h of incubation with alpha-methyl-mannoside and EDTA. Fresh suspensions contained 5--35% monocytes with an average cellular volume 2--2.5 times that of small lymphocytes. The nuclear volume of monocytes was similar to that of small lymphocytes within 10% and remained constant upon stimulation. In cultures given the optimal dose of Con A, 30 micrograms/ml, measurable cell growth commenced within 8--12 h, whereas nuclear growth began some 6 h later, which is approximately 6--8 h before increased 3H-thymidine incorporation could be detected. The average volume of responding cells grew by a factor of about 4 until mitosis, which started after around 48 h, whereas the average nuclear volume increased by a factor of about 3. The percentage of cells responding by volume growth increased with mitogen concentration even for hyperoptimal concentrations (150 micrograms/ml) which strongly suppressed 3H-thymidine incorporation. The results exemplify the type of information obtained by volume spectroscopy of leucocyte cultures. This method appears to have several advantages over conventional assays of immune response.

Histomorphic and histochemical observations on the adrenals of the reserpine-treated lizards, Calotes versicolor.

1. Cytomorphic and cytochemical studies were made on the adrenal cortex and medulla in the garden lizard, Calotes versicolor at 4, 24, 72 and 120 hr stages following a single intramuscular injection of reserpine (0.8 mg/100 g body wt.).2. The concentration of the total catecholamines in general as well as noradrenaline in particular, and the total catecholamine-containing as well as noradrenaline-containing areas became reduced at 4 and 24 hr stages, but remained similar to those in the corresponding controls at 72 and 120 hr stages. Significant alteration in the average nuclear volume did not occur at any of the selected experimental periods. These findings indicated that reserpine induced initial depletion of the stored noradrenaline (and adrenaline?) but did not lead to any remarkable activation of the adrenomedullary cells.3. The ascorbic acid concentrations in the adrenocortical cells in most or all of the experimented lizards decreased at 4 and 24 hr stages but remained along the control level of concentrations at subsequent periods (72 and 120 hr). The average nuclear volume of these cells increased significantly at 24 hr stage only. These findings indicated that reserpine stimulated adrenocortical activity that persisted at the initial experimental stages (4 and 24 hr) and subsequently attained the control level of activity.

S.N. Banerjee: &lt;i&gt;Pinus resinosa&lt;/i&gt;の発育中の

Ovules ofPinus resinosafrom 1 and 2 year-old cones were collected periodically during July to August in 1963 and 1964. The embryo-gametophyte complex containing embryos at different stages of development were irradiated by a 60Co source with doses from 0.25 to 2 kilorads. The effects of radiation on the embryo and the gametophytes were determined on the basis of abnormal differentiation and abortion of embryos and the cytological anomaly in the gametophytic cells. There was less abnormal differentiation in the embryos irradiated at stage I in comparison to other stages whereas the percentage of aborted embryos was a maximum in stage I. A possible explanation of the varying radiation effect was attempted on the basis of the average nuclear volume of embryonic cells at each stage of development. In stage I, the average nuclear volume was increased about 11 to 14-fold over those in the other stages with a concomitant peak in the percentage of aborted embryos. In contrast, higher radiation effect in root apices than that in the shoot apices could not be explained through a difference in nuclear volume. The changes in the average nuclear volume in the embryonic cells at various stages of differentiation could not be explained on the basis of an increase in the DNA content alone.

THE EFFECT OF PROTEIN DEFICIENCY ON THE DISTRIBUTION OF NUCLEAR VOLUMES IN PREPARATIONS OF RAT LIVER NUCLEI

The effect of a protein-deficient diet and the subsequent recovery period on the distribution of nuclear volumes has been studied. The results support the hypothesis that protein deficiency produces an increase in the proportion of polyploid-type nuclei in the liver as reflected in an increase in the average content of DNA per nucleus. This increase in polyploidy was shown to occur early in the experimental period, suggesting that it represents part of an adaptation mechanism more than a secondary effect produced by the deficiency of amino acids. Throughout the study, it was not possible to demonstrate any relationship between patterns of nuclear volume distribution or the average nuclear volume, and the average DNA content per nucleus. On this basis, it has been suggested that the nuclear volume might not be a unique function of the degree of ploidy, but most probably is an expression of the protein content of the organelle.

Predicted seasonal radiosensitivity of southeastern tree species

Vegetative shoot apices of 14 tree species at Oak Ridge, Tennessee were studied for 1 year (1964). The average interphase nuclear volumes, estimated interphase chromosome volumes and ratios of volumes in actively growing to dormant plants were determined. Results indicate that the predicted range in sensitivity between species for any month, approximately a 17-fold range, is constant even though the predicted sensitivity of individual species changes with respect to time.

SOME NUCLEAR CHANGES ACCOMPANYING DEVELOPMENT IN THE ENDOSPERM OF GLEDITSIA AND PODOPHYLLUM

The development of the endosperm in Gleditsia is marked by the growth of the average nucleus from 141 μ3 on June 12 to 978 μ3 on July 14. At the time of formation of a cellular endosperm the average nuclear volume commences dropping and by July 20 reaches an average of 224 μ3 throughout the endosperm. In Podophyllum at the time of cell formation the average nuclear volume begins to drop from a value of about 2850 μ3 and falls to a value of from 500 μ3 to 770 μ3, the latter estimates depending upon the mode of fixation of the material. The volume decrease in the Podophyllum endosperm nucleus is accompanied by a fall in mean relative DNA content of the average nucleus, as determined by microspectrophotometry with the two wavelength method. The possibility of degeneration of some of the higher “ploid” types is considered, as well as depolyploidization.