What's Nu(SAP) in mitosis and cancer?

Unperturbed mitosis is a prerequisite for the generation of two genetically identical daughter cells. Nucleolar-spindle associated protein (NuSAP) is an important mitotic regulator. The activity of NuSAP is essential for a variety of cellular events that occur during mitosis starting from spindle assembly to cytokinesis. In addition to playing crucial roles during mitosis, NuSAP has been in the spotlight recently due to different studies exhibiting its importance in embryogenesis and cancer. In this review, we have extensively mined the current literature and made connections between different studies involving NuSAP. Importantly, we have assembled data pertaining to NuSAP from several proteomic studies and analyzed it thoroughly. Our review focuses on the role of NuSAP in mitosis and cancer, and brings to light several unanswered questions regarding the regulation of NuSAP in mitosis and its role in carcinogenesis.     

Holokinetic composite chromosomes with “diffuse” kinetochores in the micronuclear mitosis of a heterotrichous ciliate

During micronuclear mitosis of the heterotrichous ciliate Nyctotherus ovalis Leidy rod-shaped composite chromosomes are formed by lateral association of telokinetic chromosomes. The formation of these composite chromosomes seems to be a highly ordered process since only nuclei with either 18 or 24 such chromosomes can be observed, and nuclei with the same chromosome number show a similar length distribution of their chromosomes. Further, these data indicate that we examined two otherwise indistinguishable races. During metaphase the composite chromosomes become arranged in the spindle equator in a holokinetic fashion, their entire poleward surfaces being covered by kinetochore material. These diffuse kinetochores have a trilaminar appearance comparable to those of monokinetic chromosomes. Their electron density after employing Bernhard's procedure revealed the same ribonucleoprotein distribution as reported for the localized kinetochores formed during the extranuclear mitosis in other cells. During early anaphase the outer kinetochore layer remains continuous while the individual chromosomes in the composite group show a tendency to separate leaving chromatin-free spaces of about 40 nm diameter. Kinetochore microtubules which are still anchored in the outer kinetochore layer seem to elongate and to extend into the interpolar spindle region predominantly through these holes in the chromatin. These observations suggest a like polarity of kinetochore and interpolar microtubules in the polar spindle region while microtubules in the interpolar space seem to interdigitate in an antiparallel fashion. The activity of the kinetochore to act as a microtubule-organizing center (MTOC) seems to be modulated by the chromatin underlying the outer kinetochore layer which may prevent further outgrowth of kinetochore microtubules.     

Heterochromatin protein HP1Hsβ(p25β) and its localization with centromeres in mitosis

Some autoimmune sera containing anticentromere autoantibodies also recognize a doublet of Mr 23 000 (p23) and 25 000 (p25) in addition to CENP (centromere protein)-A (Mr 19 000), -B (Mr 80 000), and -C (Mr 140 000). A p25 antigen (HP1^Hsα) has been shown to be a human homolog of Drosophila HP1 (heterochromatin protein 1). We have isolated a cDNA clone encoding another form of p25 (HP1^Hsβor p25β) from a λZap HepG2 library using human autoimmune serum. The deduced amino acid sequence of the clone contained a conserved chromodomain (chromatin modifier domain) in the N-terminal region and a heterochromatin binding domain in the C-terminal region. In immunofluorescence experiments, only affinity purified antibodies reactive with the C-terminal (amino acids 70–185) domain showed nucleoplasmic and heterochromatin staining, whereas N-terminal (amino acids 1–115) specific antibodies were nonreactive. In metaphase chromosome spreads, the C-terminal domain antibody was also localized to the centromeric regions of chromosomes. Association with centromeres was most prominent at anaphase and changed to a more generalized association with whole chromosomes in telophase. The cooccurrence of autoantibodies to centromere proteins and HP1 in certain autoimmune diseases might be a reflection of coordinated immune responses to these closely associated sets of proteins. Received: 8 August 1996; in revised form: 4 December 1996 / Accepted: 17 December 1996     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Effect of hypothermia (20–25°C) on mitosis in PtK1 cells

PtK₁ cells enter prophase and complete mitosis at 24–25°C but are inhibited from entering prophase at 20–21°C. Cells which have progressed up to midprophase at 24–37°C return to interphase when cooled to 20–21°C, but those in late prophase complete a normal, although prolonged mitosis. If prophase cells which have reverted to interphase at 20–21°C are incubated at 24–37°C they reenter prophase and complete mitosis. This temperature-induced prophase-interphase-prophase transition can be repeated several times on the same cell. At 24–25°C the process of spindle formation (i.e. prometaphase to the initiation of anaphase) encompasses approximately 75% of the total mitotic interval, with a duration of 8–12 h, compared to about 50% of the mitotic interval and a duration of 0.5 to 1.0 h at 37°C.     

A caspase-3 ‘death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical ‘death-switch'' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [¹⁸F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The ‘death-switch'' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of ‘death-switched'' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [¹⁸F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [¹⁸F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.     

Synthesis of lantadene analogs with marked in vitro inhibition of lung adenocarcinoma and TNF-α induced nuclear factor-kappa B (NF-κB) activation

The new series of pentacyclic triterpenoids reduced lantadene A (3), B (4), and 22β-hydroxy-3-oxo-olean-12-en-28-oic acid (5) analogs were synthesized and tested in vitro for their NF-κB and IKKβ inhibitory potencies and cytotoxicity against A549 lung cancer cells. The lead analog (11) showed sub-micromolar activity against TNF-α induced activation of NF-κB and exhibited inhibition of IKKβ in a single-digit micromolar dose. At the same time, 11 showed promising cytotoxicity against A549 lung cancer cells with IC₅₀ of 0.98 μM. The Western blot analysis further showed that the suppression of NF-κB activity by the lead analog 11 was due to the inhibition of IκBα degradation, a natural inhibitor of NF-κB. The physicochemical evaluation demonstrated that the lead analog 11 was stable in the simulated gastric fluid of pH 2, while hydrolyzed at a relatively higher rate in the human blood plasma to release the active parent moieties. Molecular docking analysis showed that 11 was hydrogen bonded with the Arg-31 and Gln-110 residues of the IKKβ.     

On the Roles of Heterochromatin and Euchromatin in Meiosis in Drosophila: Mapping Chromosomal Pairing Sites and Testing Candidate Mutations for Effects on X–Y Nondisjunction and Meiotic Drive in Male Meiosis

Mapping of pairing sites involved in meiotic homolog disjunction in Drosophilahas led to conflicting hypotheses about the nature of such sites and the role of heterochromatin in meiotic pairing. In the female-specific distributive system, pairing regions appear to be exclusively heterochromatic and map to broad regions encompassing many different sequences. In male meiosis, autosomal pairing sites appear to be distributed broadly within euchromatin but to be absent from heterochromatin, whereas the X-pairing site maps in the centric heterochromatin. The X site has been shown to coincide with the intergenic spacer (IGS) repeats within the rDNA arrays shared between the X and Y. It has not been clear whether the heterochromatic location of this pairing site has any significance. A novel assay for genic modifiers of X–Y chromosome pairing was developed based on the intermediate nondisjunction levels observed in males whose X chromosome lacks the native pairing site but contains two transgenic insertions of single rDNA genes. This assay was used to test several mutations in Su(var)(Suppressor of position effect variegation), PcG(Polycomb-Group) recombination defective, and repair-defective genes. No strong effects on disjunction were seen. However, the tests did uncover several mutations that suppress or enhance the meiotic drive (distorted X-Y recovery ratio) that accompanies X–Y pairing failure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water turbidity–induced alterations in coloration and courtship behavior of male guppies (Poecilia reticulata)

acta ethologica - Water turbidity deteriorates visibility and thereby may change the physiology and behavior of aquatic animals that rely on vision. In the guppy fish (Poecilia reticulata), a key...     

Effects of two species of macroalgae—Ulva pertusa and Gracilaria lemaneiformis—on growth of Heterosigma akashiwo (Raphidophyceae)

The effects of fresh thalli, culture filtrate, water-soluble extract and dry powder of two species of macroalgae, Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta), on the growth of a bloom-forming microalga, Heterosigma akashiwo, were studied in co-culture under controlled laboratory conditions. Both fresh thalli and culture filtrate of U. pertusa and G. lemaneiformis, particularly in the form of fresh thalli, significantly inhibited microalgal growth; indeed, the microalga was completely killed during the course of the experiment. A clear concentration-dependent relationship was observed between the initial concentration of fresh thalli (either U. pertusa or G. lemaneiformis) and its inhibitory effect on H. akashiwo. Simultaneous nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis fresh thalli co-culture but remained well above nutrient limitation for microalgal growth in U. pertusa co-culture, in which the microalgal cells were completely killed. However, daily f/2 medium repletion would obviously alleviate the growth inhibition in G. lemaneiformis co-culture. Since the present study was carried out under controlled conditions, fluctuations in environmental factors (i.e., light, temperature, carbon limitation, bacterial presence and pH) were limited during the experiment. We thus concluded that allelopathy was the most likely explanation for microalgal growth inhibition in U. pertusa co-culture, while the combined roles of allelopathy and nutrient limitation were responsible for growth inhibition in G. lemaneiformis co-culture. Similarly, macroalgal water-soluble extracts and dry powders affected the co-cultured H. akashiwo greatly, with more obvious effects observed in water-soluble extract co-cultures. A dose-dependent relationship was also observed over the course of the experiment. It can be concluded that macroalgal thalli contain some bioactive compounds. The results of the present study suggest that U. pertusa and G. lemaneiformis, especially in the form of fresh thalli, effectively inhibit the growth of H. akashiwo and could thus be potential candidates for use in the control and mitigation of H. akashiwo blooms.     

Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

Parkinson’s disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein α-synuclein (α-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and α-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between α-syn and Fe(II) (5.8 × 10³ M⁻¹) has been measured, but studies on the binding between α-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between α-syn and Fe(II) and the redox property of the resultant α-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O₂) to the putative α-syn-Fe(III) complex, with H₂O₂ as a co-product. The reduction potential was estimated to be 0.025 V vs. Ag/AgCl, which represents a shift by −0.550 V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between α-syn and Fe(III), 1.2 × 10¹³ M⁻¹, to be deduced. Despite the relatively high binding affinity, α-syn-Fe(III) generated from the oxidation of α-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)₃ and/or ferrihydrite gel. The roles of α-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed α-syn aggregation, and iron transfer processes.     

Estimation of relative contribution of “mobile phycobilisome” and “energy spillover” in the light–dark induced state transition in Spirulina platensis

Previously, it was clarified that phycobilisome (PBS) mobility and energy spillover were both involved in light-to-dark induced state transitions of intact Spirulina platensis cells. In this work, by taking advantage of the characteristic fluorescence spectra of photosystem I (PSI) trimers and monomers as indicators, the relative contributions for the “mobile PBS” and “energy spillover” are quantitatively estimated by separating the fluorescence contribution of PBS mobility from that of PSI oligomeric change. Above the phase transition temperature (T _PT) of the membrane lipids, the relative proportion of the contributions is invariable with 65% of “mobile PBS” and 35% of “energy spillover”. Below T _PT, the proportion for the “mobile PBS” becomes larger under lowering temperature even reaching 95% with 5% “energy spillover” at 0°C. It is known that lower temperature leads to a further light state due to a more reduced or oxidized PQ pool. Based on the current result, it can be deduced that disequilibrium of the redox state of the PQ pool will trigger PBS movement instead of change in the PSI oligomeric state.