Nucleoplasm Research Articles

Ultra-Wideband Impedance Spectroscopy of the Nucleus in a Live Cell

This paper describes the first application of a double-shell model in ultra-wideband impedance spectroscopy of a live cell. The impedance spectrum measured from 9 kHz to 9 GHz is analyzed by using the double-shell cell model to separate the contribution of the nucleus from that of the cytoplasm. The model parameters include the nucleus-to-cell radius ratio (r) and a parallel R-C circuit for each of the four cell compartments: the cell membrane (CM), the cytoplasm (CP), the nuclear membrane (NM), and the nucleoplasm (NP). In general, the extracted radius ratio agrees with that measured by optical microscopy, and the extracted resistances and capacitances agree with the literature and order-of-magnitude estimates. For example, for a human lymphocytes cell with r ≈ 0.8, the nuclear contribution is approximately linear. Therefore, the membrane parameters are constant so that RCM = 1.5 MΩ, CCM = 1.5 pF, RNM = 0.05 MΩ, and CNM = 1.2 pF, but the plasma parameters are linearly scaled so that RCP → 0.5(1 − r) MΩ, CCP → 6.7/(1 − r) fF, RNP → 0.12r MΩ, and CNP → 9.4/r fF. Because morphological and structural changes of a cell nucleus are important screening, diagnostic, and prognostic markers, these results suggest that ultra-wideband impedance spectroscopy may be a fast, compact, and label-free technique in cancer cytology.

Do the Cytoplast and Nuclear Material of Germinal Vesicle Oocyte Support Developmental Competence Upon Reconstruction with Embryonic/Somatic Nucleus.

Maturation conditions and oocytes quality have substantial roles on developmental competence of unreconstructed or reconstructed oocytes. Cloning has been reported successfully with low efficiency through embryonic or somatic nuclear transfer into enucleated metaphase II oocytes. It has been suggested that introducing embryonic or somatic nucleus to cytoplast at earlier stage might improve reprogramming of the introduced nucleus. In addition, the synchronization between the donor nucleus and recipient cytoplasts might effect on reprogramming and further embryonic development. Therefore, the question arises; does the cytoplast of germinal vesicle (GV) oocyte containing nuclear sap improve developmental competence upon reconstruction with embryonic/somatic nucleus compared with MII cytoplast. It has been indicated that GV material is essential for remodeling of sperm or embryonic or somatic nucleus in GV oocyte cytoplast and their further embryonic developmental competence. GV cytoplasts could be obtained through micromanipulation. Different micromanipulation techniques of immature oocytes at different stages were adapted in addition to introducing donor nuclei at G0/G1, S and G2/M phase, and enucleolation technique as well. Upon micromanipulation, it could obtain GV cytoplasts; cumulus-free and without GV material, cumulus-complexes and without GV material, cumulus-free and with GV material, and cumulus-complexes and with GV material in addition to enucleolated GV oocytes. Therefore, this short review will address briefly the importance of maturation conditions, cumulus cells, oocyte quality, the techniques of enucleation GV oocyte, the cell cycle stage of the introduced donor cell, or nucleus for oocyte maturation and embryo development.

Progesterone Receptor Membrane Component-1 (PGRMC1) nucleolar localization in bovine granulosa cells and its putative interaction with nucleolin.

PGRMC1 is a multifunctional protein that is found in multiple subcellular compartments, suggesting a specific function at each site. Among the several subcellular sites of expression, PGRMC1 was found in the nucleolus of human cells (Ahmad et al. 2009) and bovine zygotes (Luciano et al. 2010). However, the role at this site is not clear. Therefore, the aim of this study is to assess whether PGRMC1 modulates nucleolar function. Immunofluorescence experiments confirmed nucleolar localization in rat spontaneously immortalized granulosa cells, bovine granulosa cells (bGC) and bovine oocytes. Moreover, in bGC PGRMC1 co-localizes with nucleolin, a well-known nucleolar marker exerting important functions within the nucleolus. Additionally, siRNA mediated gene knockdown experiments showed that when PGRMC1 expression is silenced, nucleolin localization shifts from the nucleolus to the nucleoplasm, suggesting a PGRMC1/nucleolin functional association. However, in situ proximity ligation assay did not detect a direct interaction between these two proteins, suggesting the involvement of additional molecules that could mediate PGRMC1/nucleolin interaction. In conclusion, these studies suggest a function for PGRMC1 in nucleolar activity and set the stage for further investigations aimed at dissecting PGRMC1’s molecular mechanisms of action in the nuclear compartment.

AB091. Analysis of LA2 as a functional candidate gene for Emery Dreifuss muscular dystrophy and dilated cardiomyopathy.

Laminopathies are rare genetic disorders caused by mutations in genes encoding lamins or lamin-interacting proteins. LMNA, EDM and to a lower measure the Nesprin genes have been associated with Emery Dreifuss muscular dystrophy (EDMD), characterized by contractures of the Achilles tendons, progressive skeletal muscle weakness and heart rhythm disturbances leading to dilated cardiomyopathy (DCM) and sudden cardiac death. Since ~60% EDMD patients are not associated to known genes, we used a functional candidate-gene approach to identify additional EDMD disease genes. Based on reported interactions of lamina associated polypeptide 2 (LAP2) with nucleoplasmic lamin A/C and the association of the LAP2alpha isoform to DCM (Matthew R.G Taylor et al. 2005), 111 EDMD and 87 DCM patients were investigated for DNA variations in LAP (encoding six LAP2 isoforms) using heteroduplex analysis and direct sequencing. Among ten variations found, four changes—p.P426L (c.1481C>T) in LAP2alpha, p.D271E (c.1054T>G) in LAP2beta, and p.V423L (c.1058G>C) and p.M381I (c.1387 G>A) in LAP2gamma—were unique for EDMD patients, but segregation analysis indicated only p.P426L LAP2alpha as a mutation potentially associated to EDMD. P426L-LAP2 alpha localized to the nucleoplasm in skin fibroblasts, like wild-type protein and did not affect the loalization of lamin A. However, in mutant fibroblast cultures phosphorylated retinoblastoma protein (Rb) levels were reduced compared to wild-type cultures, sugesting that the mutated protein may affect cell cycle progression in agreement with previous studies implicating LAP2alpha-lamin A in Rb-mediated cell cycle control. The present study shows that LAP2alpha mutations might add to the pathology of EDMD in ~1% of EDMD patients.

Molecular Characterization of UKp83/68, a Widespread Nuclear Proteins that Bind Poly(A) and Colocalize with a Nuclear Speckle's Component.

We have cloned a gene from a rat liver cDNA library, representing alternatively spliced cDNAs encoding 83-kDa and 68-kDa proteins, which we have designated as UKp83 and UKp68, respectively. Both proteins have a predicted nuclear localization signal and five CCCH motifs (zinc-binding motifs), and share a degree of sequence similarity with Nab2, a yeast protein that contains nucleic acidbinding motifs and tandem CCCH zinc fingers. Nab2 binds homopolymeric RNA and single-stranded DNA and regulates poly(A) tail length and the export of mRNA to the cytosol. The CCCH motifs of UKp83/68 bound poly(A) and ssDNA strongly and other RNA homopolymers and dsDNA less efficiently. The UKp83/68 protein localized within the nucleus with a fibrous or punctate structure that reflected the distribution of SC35, a known marker of nuclear speckles which are nuclear domains enriched in pre-mRNA splicing factors and located in the interchromatin regions of the nucleoplasm of mammalian cells. The distribution of UKp83/68 changed during the different stages of mitosis. During prometaphase, when the nuclear envelope disintegrates, the protein becomes partially localized on the chromosomes; at other times, transiently dispersed over the cytoplasm with the formation of fibrous structure. The transient expression of UKp83 in HEK293T cells had no apparent effect on cellular function, whereas the expression of an antisense sequence or C-terminal domain of UKp83 induced apoptosis. These results suggest that UKp83/68 is probably essential for cell viability and may play important role in mRNA processing.

Relationship between ultrastructure, expression of MMP-9 and VEGF and invasiveness in pituitary adenomas

Objective To study the relationship between the ultrastructural characteristics,MMP-9 and VEGF and invasiveness in pituitary adenomas.Methods To observe the difference between the two groups of pituitary adenomas (30 cases in invasive group,30 cases in non-invasive group) in the ultrastructural characteristics by electron microscope,including the rough endoplasmic reticulum,the number of mitochondria,ribosomes,nuclear atypia and nucleolus,and the immunohistochemistry result to analyse the relationship between the ultrastructural characteristics,expression of MMP-9 and VEGF and invasiveness in pituitary adenomas.Results The rough endoplasmic reticulum is increased,the ribosome is rich,the nuclei is irregular in shape and the nucleolu is huge in the cytoplasm of invasive pituitary adenomas,and the nucleolus occurrence rate,nuclear atypia rate,the nucleoplasm ratio and the number of mitochondrias in invasive pituitary adenomas were higher than those in the non-invasie group (P ＜ 0.01).MMP-9 and VEGF show strong positive expression in invasive pituitary adenomas.Conclusions Uhrastructural characteristics are related with the invasiveness of pituitary adenomas. Key words: Invasive pituitary adenomas ; Ultrastructural characteristics ; Matrix metalloproteinase-9; Vascular endothelial growth factor

Amplification of ribosomal DNA in Acheta. VII. Transfer of DNA-RNA assemblies from the nucleus to the cytoplasm.

In the cricket Acheta the major chromomere of chromosome 6 is at late pachytene the main site of amplification of ribosomal DNA. As meiosis progresses into late diplotene, two types of nucleolar structures can be recognized: the main and the secondary nucleolar components. The latter are few and are easily distinguishable from the first type by their larger size, spherical shape and the presence of three zones. Both nucleolar components contain DNA. The DNA of the main nucleolus has been shown to be ribosomal DNA. The two types of nucleolar components are seen, in the light and electron microscopes, to migrate to the nuclear sap and later to the nuclear envelope. These large DNA-RNA assemblies move out of the nucleus into the cytoplasm, in an undisrupted form, by pushing the nuclear envelope and building a vesicle surrounded by the nuclear membrane. This vesicle later separates from the nucleus and the DNA-RNA assembly appears free in the cytoplasm. The transfer of a specific type of DNA, by intracytosis, to the cytoplasm at a period preceding fertilization is of particular genetic significance.

Dppa2 and Dppa4 Are Closely Linked SAP Motif Genes Restricted to Pluripotent Cells and the Germ Line

Despite the enormous medical potential of ESCs, the molecular mechanisms conferring the ability to differentiate into all cell types of the embryo remain elusive. We used an in silico approach to identify genes expressed exclusively in mouse preimplantation embryos and pluripotent cell lines. Two of these genes were developmental pluripotency-associated gene 2 (Dppa2) and Dppa4, which we show are closely linked genes encoding putative nuclear SAP domain proteins expressed in human and mouse pluripotent stem cells and germ cell tumor-derived embryonal carcinoma cells. In the mouse, these genes are transcribed in germinal vesicle-stage oocytes and throughout the cleavage stages of embryogenesis. They then become restricted to the pluripotent inner cell mass of blastocysts and are subsequently downregulated. After gastrulation, Dppa2 and Dppa4 are expressed only in the developing germ line, showing that these genes mark cells of the pluripotent cycle. In the germ line, both genes are downregulated as the germ cells commit to the oogenic pathway or soon after commitment to the spermatogenic pathway. We have observed similar germ line expression profiles for other pluripotent markers, and these results are consistent with the hypothesis that pluripotent markers must be downregulated during fetal germ line development, a process that may be required to facilitate appropriate germ line differentiation. The study of expression and function of pluripotent markers such as Dppa2 and Dppa4 is likely to unveil new aspects of the regulation of pluripotency and germ line development in mammals.

VP-16 (etoposide) and calphostin C trigger different nuclear but akin cytoplasmic patterns of changes in the distribution and activity of protein kinase C-βI in polyomavirus-transformed pyF111 rat fibroblasts

Protein kinase C (PKC) isoforms regulate cell proliferation and apoptosis. Since the PKC isoenzyme complement varies considerably from cell type to cell type, a PKC's responsiveness to an apoptogenic challenge must be defined for both the type of apoptogen and the type of cell. We have already reported that the changes in the distribution and activity of PKC-delta in apoptosing polyomavirus-infected/transformed Fischer rat embryo pyF111 fibroblasts depend on the type of apoptogen. Here, we show that this is also true for PKC-betaI in pyF111 cells treated with the slow DNA-damaging VP-16 (etoposide) or the fast-acting (in the cytoplasm) calphostin C. These apoptogens caused quite different shifts of the PKC-betaI level and activity in the nuclear membrane (NM) and nucleoplasm (NP), but corresponding changes in the cytosol (CS) and cytoplasmic particulate (CP) fractions. The hefty translocation of PKC-betaI onto the CP fraction and its increased activity there suggest the possible triggering of a cytochrome c/caspase-mediated apoptosis-inducing mechanism common to both agents. The present results are a necessary lead-up to functional proteomic analyses aimed at identifying the molecules forming the local PKC-betaI signalling modules under different conditions.

Influence of breast carcinoma cells on normal endothelial cells: experimental study with a co-culture system

To study the influence of breast carcinoma cells on normal endothelial cells. Human endothelial cells were isolated from umbilical cord blood. Medium Z-MCF-7-EC was established and was used to co-culture the normal endothelium cells (ECs) and human breast cancer cells of the line MCF-7. Normal endothelial cells cultured by itself were used as control. Light microscopy and transmission electron microscopy were used to observe the morphology of the 2 kinds of endothelial cells. Expression of the genes ESM, IGFBP-3, alphavbeta3, VE-C, and Tie-2-2 was analyzed by semi-quantitative RT-PCR using a house-keeping gene beta(2m) as inner reference gene. The ECs co-cultured with MCF-7 breast cancer cells were abnormal in shape with increased size of nucleus and nucleolus, increased size ratio of nuclear to nucleoplasm, increased depth of surface fenestration, loosed and distorted endoplasm, increased size of cell-cell junctions, decreased number of surface microvilli, and tubules formed by ECs. The expression values of the genes ESM, IGFBP-3, alphavbeta3, VE-C, and Tie-2-2 in the ECs co-cultured with MCF-7 breast cancer cells were all up-regulated in comparison with those in the controls (P < 0.01, P < 0.05, P < 0.01, P < 0.01, and P < 0.05 respectively). Breast cancer cells promote formation of new vessels with endothelial cells different from the normal ECs in character and behavior.

Post-transcriptional polyadenylation is probably an essential step in selection of Balbiani ring transcripts for a cytoplasmic role.

Some aspects of poly(A) addition on to the RNA of the two major Balbiani rings 1 and 2 and of the fate of polyadenylated and nonpolyadenylated 75-S RNA transcripts in salivary gland cells of Chironomus tentans have been investigated. RNA derived from the Balbiani ring portion of chromosome IV, essentially representing a spectrum of nascent growing transcripts, lacks poly(A) segments, while about 16% of the finished 75-S RNA molecules released into the nuclear sap is associated with poly(A) stretches. More than 90% of the recently arrived cytoplasmic 75-S RNA molecules contain poly(A) chains that consist of about 103 AMP residues. Poly(A)-containing 75-S RNA rapidly disappears from the nucleus and no significant amount of nonpolyadenylated Balbiani ring RNA molecules can be chased into the cytoplasm. A large proportion of Balbiani ring transcripts remains without poly(A) segments in the nuclear sap even after prolonged incubation periods and probably never leaves the nucleus. It seems probable therefore that the molecular mechanism operating in the selection of 75-S RNA chains for cytoplasmic use includes post-transcriptional poly(A) addition as an obligatory event in the biogenesis of Balbiani ring RNA. Another defined mRNA fraction of non-Balbiani-ring origin, designated 35-S RNA, which emerges in the nuclear sap after 45 min of labelling with tritiated RNA precursors, appears to be polyadenylated up to 100% and becomes to a large extent converted to cytoplasmic 35-S RNA. A small but significant proportion, about 13%, of hnRNA produced by non-Balbiani-ring genes on the chromosomes I–III is polyadenylated. The corresponding figure for hnRNA in the nuclear sap is about 25%, and for mRNA in the cytoplasm 56%.

Cellular localization of the apurinic/apyrimidinic endodeoxyribonucleases in rat liver.

A method has been developed to purify rat liver nuclei; the isolated nuclei keep both nuclear membranes and retain more than 90% of the cell apurinic/apyrimidinic (AP) endodeoxyribonuclease activity. The nuclear enzyme is located mostly in chromatin non-histones; there is also an important amount of activity in the nuclear sap and some in the nuclear membranes. The cytoplasmic AP endodeoxyribonuclease activity is shared between mitochondria, cytosol and membranes. Different cell compartments appear to contain different AP endodeoxyribonuclease species: the membrane enzyme is activated by Triton whereas the other enzymes are rather inhibited; the nuclear sap enzyme has a higher molecular weight and a higher thermal resistance than the chromatin enzyme. A hypothesis is formulated according to which: (1) the chromatin enzyme is the only species important for nuclear DNA repair; (2) the species present in the other cell compartments might be precursors of the chromatin AP endodeoxyribonuclease.

LRP130, a single-stranded DNA/RNA-binding protein, localizes at the outer nuclear and endoplasmic reticulum membrane, and interacts with mRNA in vivo

LRP130 (also known as a LRPPRC) is an RNA and single-stranded DNA-binding protein, and recently identified as a candidate gene responsible for the Leigh syndrome, a French-Canadian type cytochrome c oxidase deficiency. However, the biological function of LRP130 still remains largely unresolved. In the present study, we found that the C-terminal half of the mouse LRP130 located within a 120 amino acid sequence (a.a. 845–964) binds to synthetic RNA homopolymers, poly(G), poly(U), and poly(C), as well as r(CUGCC) 6. Assessment of the subcellular localization indicated both nuclear/endoplasmic reticulum (ER) and mitochondrial fractions to be positive. To further analyze the subcellular localization of LRP130, a nuclear/ER fraction was fractionated into the nucleoplasm (NP) and nuclear envelope (NE)/ER, and the latter was further separated into outer nuclear membrane (ONM)/ER and inner nuclear membrane (INM) by treatment with Triton X-100. LRP130 was detectable in all three fractions, and the distribution pattern was in good accordance with that known for ONM/ER proteins. Interestingly, immunostaining of HeLa cells demonstrated nuclear rim staining of LRP130, specifically at the outside of the NE and also at ER, and association of LRP130 with poly(A) + RNA was restricted only to the ONM/ER fraction. Overexpression of full-length mouse LRP130 fused with EGFP resulted in nuclear accumulation of poly(A) + RNA in HeLa cells. Taking all these results together, it is suggested that LRP130, a novel type of RNA-binding protein, associates with mRNA/mRNP complexes at the outside of NE and ER, and plays a role in control of mRNA metabolisms.

Expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in pituitary tumours.

The present study was performed to investigate HIF-1alpha (hypoxia-inducible factor-1alpha) expression in a large number of immunohistochemically and ultrastructurally characterized surgically removed pituitary tumours. The potential relation of HIF-1alpha with outcome variables as well as the presence of HIF-1alpha expression in the tumours treated with dopamine agonists and octreotide, a long-acting somatostatin analogue was also investigated. HIF-1alpha immunoreactivity was confined to the nucleoplasm whereas the nucleoli were unconspicuous. The distribution of HIF-1alpha was evident in the tumours whereas normal adenohypophysial cells showed no HIF-1alpha staining. HIF-1alpha expression was detected not only in the tumour cells but also in endothelial cells lining the blood vessels within the tumour. ACTH producing adenomas showed the lowest level of HIF-1alpha expression whereas pituitary carcinomas and GH producing adenomas had the highest counts. The statistical study demonstrated no significant correlation between HIF-1alpha expression, patient age, gender, tumour, size, invasiveness, cell proliferation rate and vascularity. These results suggest that the behaviour of pituitary tumours does not primarily depend of HIF-1alpha expression. Our study demonstrated an increase HIF-1alpha expression in bromocriptine treated PRL producing pituitary adenomas compared with untreated tumours but no increase in octreotide treated tumours.

Dynamic continuity of nuclear and mitotic matrix proteins in the cell cycle.

The eukaryotic cell nucleus is a membrane-enclosed compartment containing the genome and associated molecules supported by a highly insoluble filamentous network known as the nucleoskeleton or nuclear matrix. The nuclear matrix is believed to play roles in maintaining nuclear architecture and organizing nuclear metabolism. Recently, advances in microscopic techniques and the availability of new molecular probes have made it possible to localize functional domains within the nuclear matrix and demonstrate dynamic interactions between both soluble and insoluble components involved in the control of multiple nuclear transactions. Like the cytoplasm and its skeleton, the nucleoplasm is highly structured and very crowded with an equally complex skeletal framework. In fact, there is growing evidence that the two skeletal systems are functionally contiguous, providing a dynamic cellular matrix connecting the cell surface with the genome. If we impose cell cycle dynamics upon this skeletal organization, it is obvious that the genome and associated nuclear matrix must undergo a major structural transition during mitosis, being disassembled and/or reorganized in late G2 and reassembled again in daughter nuclei. However, recent evidence from our laboratory and elsewhere suggests that much of the nuclear matrix is used to form the mitotic apparatus (MA). Indeed, both facultative and constitutive matrix-associated proteins such as NuMA, CENP-B, CENP-F, and the retinoblastoma protein (Rb) associate within and around the MA. During mitosis, the nuclear matrix proteins may either become inert "passengers" or assume critical functions in partitioning the genome into newly formed G1 nuclei. Therefore, we support the view that the nuclear matrix exists as a dynamic architectural continuum, embracing the genome and maintaining cellular regulation throughout the cell cycle.

Evaluation of the sensitivity of the terminal deoxynucleotidyl transferase-immunogold technique on Balbani ring genes.

Recently, we developed the terminal deoxynucleotidyl transferase (TdT)-immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were evaluated using the giant polytene chromosomes from Chironomus tentans salivary glands. Emphasis was put on the Balbiani rings (BRs), specialized chromosomal sites with exceptionally intense synthesis of large mRNA molecules. Immunolabeling was recorded not only over the bands and interbands of the polytene chromosomes but also over the BR structures. In the BRs, gold particles were present over segments of active transcription units, each with a central chromatin axis and a number of growing RNP products attached to the axis. One third of the transversely sectioned transcription units showed labeling in the central parts, i.e., where the unfolded chromatin axis is located, whereas the growing RNP fibers remained unlabeled. The absence of labeling of the RNP fibers is not likely to be due to lack of accessibility, because anti-RNA antibodies readily decorated the RNP fibers. The nuclear sap and cytoplasm displayed no significant label. These results clearly indicate that the TdT-immunogold technique is specific for DNA and detects not only DNA in compacted chromatin but also fully extended DNA. Its ability to efficiently label a single DNA molecule demonstrates the method's very high sensitivity.

MULTIPLE PATHWAYS FOR APOPTOTIC NUCLEAR FRAGMENTATION

We analyzed the ultrastructure of apoptotic nuclear fragmentation in U937 cells treated with many different apoptogenic agents. We found that this characteristic apoptotic feature can be achieved through multiple alternative pathways, depending on the apoptogenic inducer, leading to slightly different final nuclear morphologies. In most instances, the irregularly shaped nucleus of U937 rounds up; then, chromatin condenses at the nuclear periphery. Condensed chromatin can form protruding patches, which eventually bud from the nucleus in sealed vesicles through a process which is actin-dependent, since it could be blocked by cytochalasins. Alternatively, chromatin condenses in tiny, nonprotruding crescents, and a cleavage in the nuclear sap forms, beginning from the inner nuclear membrane and growing inward, thus splitting the nucleus. In U937 induced to apoptosis by hydrogen peroxide in the presence of ADP-ribosylation inhibitors, the nuclei fragment in many vesicles before chromatin even begins to condense: chromatin condensation probably occurs as a consequence. While all the apoptotic morphologies described above evolve from interphase cells, a peculiar apoptotic morphology, possibly deriving from mitotic cells, is detected upon oxidative stress, recalling the formation of micronuclei by clastogenic treatments; it shows partially membrane-bound chromatin patches, which look midway between condensed chromosomes and apoptotic condensed chromatin. The existence of these multiple pathways for nuclear fragmentation may indicate an evolutionary convergence, suggesting that this event may play an important physiological role in apoptosis.

Multiple Pathways for Apoptotic Nuclear Fragmentation

We analyzed the ultrastructure of apoptotic nuclear fragmentation in U937 cells treated with many different apoptogenic agents. We found that this characteristic apoptotic feature can be achieved through multiple alternative pathways, depending on the apoptogenic inducer, leading to slightly different final nuclear morphologies. In most instances, the irregularly shaped nucleus of U937 rounds up; then, chromatin condenses at the nuclear periphery. Condensed chromatin can form protruding patches, which eventually bud from the nucleus in sealed vesicles through a process which is actin-dependent, since it could be blocked by cytochalasins. Alternatively, chromatin condenses in tiny, nonprotruding crescents, and a cleavage in the nuclear sap forms, beginning from the inner nuclear membrane and growing inward, thus splitting the nucleus. In U937 induced to apoptosis by hydrogen peroxide in the presence of ADP-ribosylation inhibitors, the nuclei fragment in many vesicles before chromatin even begins to condense: chromatin condensation probably occurs as a consequence. While all the apoptotic morphologies described above evolve from interphase cells, a peculiar apoptotic morphology, possibly deriving from mitotic cells, is detected upon oxidative stress, recalling the formation of micronuclei by clastogenic treatments; it shows partially membrane-bound chromatin patches, which look midway between condensed chromosomes and apoptotic condensed chromatin. The existence of these multiple pathways for nuclear fragmentation may indicate an evolutionary convergence, suggesting that this event may play an important physiological role in apoptosis.

Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes.

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

Aluminium and Alzheimer's disease: sites of aluminium binding in human neuroblastoma cells determined using 26Al and accelerator mass spectrometry

The aluminium distribution between the major cell compartments of human neuroblastoma cells grown in culture has been determined using 21Al and accelerator mass spectrometry (AMS). Cells (IMR-32) were grown for eight days in a culture medium containing Al-EDTA (0.2mM) spiked with 26Al, harvested, and fractionated by standard biochemical techniques. 26Al in fractions after ashing to Al 2O 3 was determined by AMS using the 14UD accelerator at ANU Canberra. The cytoplasmic and nuclear cell compartments appeared to have reached diffusive equilibrium with the culture medium. Whilst 26Al was retained by the nuclear proteins and nuclear sap, 26Al did not appear to bind to the nucleic acids (DNA/RNA).