Studying the relationship between cell cycle and Alzheimer's disease by gold nanoparticle probes

In this study, a simple gold nanoparticle (GNP)-based colorimetric assay has been developed for studying the relationship between cell cycle and β-amyloid peptide (Aβ, the biomarker of Alzheimer's disease [AD]) expression level. It was found that Aβ expression of neuronal cells (e.g., SHG-44 cell line) is strongly dependent on cell cycle phases; that is, the Aβ expression level was highest when cells were arrested in the G1/S phase by thymidine and was lowest when they were arrested in the G2/M phase by nocodazole. This finding may improve the understanding of AD pathology and provide a new tool for anti-dementia drug development.     

Evidence for a relationship between chronotype and reproductive function in women

There is evidence for the reciprocal interaction between circadian oscillation and reproduction, and disruption of circadian rhythms has been associated with impaired menstrual functions and reduced fertility in women. However, only little information is available on the relationship between reproduction and chronotype. The aim of the present study is to better assess this relationship. The participants (aged 25 to 74?yrs) were selected randomly from the Finnish Population Information System. The data from 2672 female participants of the National FINRISK Survey 2007 were analyzed to test the associations between chronotype (morning, intermediate, or evening) and reproductive features. Of the participants, 139 (5.6%) were evening, 1217 (48.7%) intermediate, and 1145 (45.8%) morning chronotypes. Among the participants aged 25 to 54?yrs, the duration of menstrual cycle was longer among evening chronotypes (28.8?±?4.4?d) than among morning (27.7?±?2.6?d; p?<?0.01) and intermediate (27.8?±?3.3?d; p?=?0.05) chronotypes. Significant correlations were found between the higher morningness-eveningness scores (the more of morning chronotype) and the shorter durations of menstrual bleeding, both in the whole sample (p?<?0.001) and after limiting the analyses to women younger than 55?yrs (p?<?0.05). In multivariable analyses on the whole sample, as compared with morning chronotypes, intermediate chronotypes had a significantly longer duration of menstrual bleeding (B?=?0.160, 95% confidence interval [CI]?=?0.044 to 0.276; p?<?0.01) as well as a higher odds for difficulties in getting pregnant (odds ratio [OR]?=?1.464, 95% CI?=?1.118 to 1.917; p?<?0.01). Our findings suggest that chronotype is related to the reproductive function in women.     

Pbcrk-1, the Plasmodium berghei orthologue of P. falciparum cdc-2 related kinase-1 (Pfcrk-1), is essential for completion of the intraerythrocytic asexual cycle

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 (pfcrk-1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P. berghei orthologue of pfcrk-1, but were unable to recover P. berghei parasites with a disrupted pbcrk-1 locus. In contrast, an integration event at this locus that did not result in a loss-of-function of the pbcrk-1 gene was readily observed. This strongly suggests that a functional pbcrk-1 gene product is essential to intraerythrocytic asexual multiplication.     

Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min⁻¹) and was decreased during the steady-state responses to insulin (0.18 min⁻¹), AICAR (0.16 min⁻¹), and A-769662 (0.24 min⁻¹). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.     

Human EML4, a novel member of the EMAP family, is essential for microtubule formation

Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.     

Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study, we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2, NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into beta-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs.     

SSU rDNA sequence support for a close relationship between the elaphomycetales and the Eurotiales and Onygenales

Based on parsimony analyses of eight new SSU rDNA sequences and 24 homologous sequences retrieved from the DNA databases, we suggest a possible phylogenetic relationship of Elaphomycetales with Eurotiales and Onygenales. Our three includedElaphomyces sequences strongly cluster together (bootstrap value 100%) within a monophyletic group (100%) of Elaphomycetales, Eurotiales, and Onygenales. Earlier reports that another cleistothecial lineage (Erysiphe) is related to Leotiales, are supported by our discovery that also another cleistothecial species,Amylocarpus encephaloides, shows affinity to Leotiales. Ascosphaeraceae and Eremascaceae are possibly better accommodated in Onygenales. We describe a new DNA extraction method in which sonication is used to disrupt thick-walled spores. It is useful for both fresh and dried fungal material.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15°C to 34°C or decreased from 39°C to 15°C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34°C swam at 570 μm/s. On incubation at 15°C these cells swam at 100 μm/s. When the temperature was increased to 34°C after a 90-min incubation at 15°C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34°C-grown cells decreased to 210 μm/s, and the cells ceased to move when the temperature was decreased to 15°C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15°C increased gradually during incubation at 15°C. On the other hand, the fluidity of the heated cell decreased during incubation at 34°C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 μm/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Double fertilization inEphedra trifurca,a non-flowering seed plant: The relationship between fertilization events and the cell cycle

Summary Fertilization inEphedra trifurca was examined with a combination of light and fluorescence microscopy. Developmental analysis clearly indicates that double fertilization events, similar to those described inE. nevadensis, regularly occur during the process of sexual reproduction inE. trifurca. In addition to the typical fusion of a sperm nucleus and egg nucleus, a second fertilization event occurs between the second sperm nucleus from an individual pollen tube and the ventral canal nucleus. Both of the fertilization events take place within the confines of an individual egg cell of the female gametophyte. Microspectrofluorometric data demonstrate that each nucleus involved in a sexual fusion event proceeds through the synthesis phase of the cell cycle and increases its DNA content from 1C to 2C before the process of nuclear fusion is completed. Photometric data also confirm that the product of the second fertilization event is equal in DNA content (4C) to the zygotic nucleus derived from the first fertilization event, and is prepared to enter into mitosis as a fully functional diploid nucleus.Abbreviations DAPI 4;,6-diamidino-2-phenylindole - RFU relative fluorescence units     

Evidence for a quinone binding site close to the interface between NUOD and NUOB subunits of Complex I

Piericidin, rotenone and pyridaben are specific inhibitors of the NADH-ubiquinone oxidoreductase (Complex I) that bind to its ubiquinone binding site(s). Using site directed mutagenesis, we demonstrate that residues G409, D412, R413 and V407 of the C-terminus of Complex I NUOD subunit are directly involved in the binding of these inhibitors. We propose that the corresponding inhibitor/quinone binding site would be located close to NUOD-NUOB interface.     

Evidence for the connections between a clutch cell and a corticotectal neuron in area 17 of the cat visual cortex

Evidence is presented for the synaptic connectivity between a physiologically characterized and intracellularly filled GABAergic interneuron and a corticotectal pyramidal neuron in area 17 of the cat visual cortex. The interneuron was located in layer 4 and had the morphological characteristics of a clutch cell. The physiological data demonstrated that the clutch cell received direct X-type innervation from the dorsal lateral geniculate nucleus. These results indicate that a GABAergic neuron is directly involved during the first cortical stages of geniculocorticotectal interactions. Furthermore, the proximal location of the clutch-cell inputs to the labelled dendrite suggests a strategic siting of intracortical feedforward inhibition.     

A Novel Interaction between the SH2 Domain of Signaling Adaptor Protein Nck-1 and the Upstream Regulator of the Rho Family GTPase Rac1 Engulfment and Cell Motility 1 (ELMO1) Promotes Rac1 Activation and Cell Motility

Nck family proteins function as adaptors to couple tyrosine phosphorylation signals to actin cytoskeleton reorganization. Several lines of evidence indicate that Nck family proteins involve in regulating the activity of Rho family GTPases. In the present study, we characterized a novel interaction between Nck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that the Nck-1-ELMO1 interaction is mediated by the SH2 domain of Nck-1 and the phosphotyrosine residues at position 18, 216, 395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which the SH2 domain was inactive), or a 4YF mutant of ELMO1 lacking these four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitor treatment and overexpression of Src family kinase Hck significantly enhanced Nck-1-ELMO1 interaction. Moreover, wild type Nck-1, but not R308K mutant, significantly augmented the interaction between ELMO1 and constitutively active RhoG (RhoG^V12A), thus promoted Rac1 activation and cell motility. Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulating Rac1 activity.     

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or , -dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.Abbreviations and symbols CHI cycloheximide - GTC guaniline thiocyanate - mt ⁺/mt ^- mating type plus or minus - PAS Periodic-acid-Schiff reagent - SDS sodium dodecyl sulphate     

Chaetal arrangement provides no support for a close relationship of Sabellidae and Sabellariidae (Annelida)

Sabellid and sabellariid polychaetes are regarded as sister groups in a number of recent phylogenetic analyses. This is based mainly on a shared specific arrangement of chaetae referred to as chaetal inversion. Remarkably, the uncini have a notopodial position in the abdomen, whereas capillary chaetae occur in the neuropodia in both taxa in contrast to the situation in putative relatives. However, in sabellids uncini and capillary chaetae change their position completely at the border between thorax and abdomen, whereas uncini are missing in the parathorax of Sabellariidae. Due to this difference the significance of the chaetal inversion for systematics has been subject to discussion for years. Serial semithin sections of parapodia of the Sabellidae Sabella pavonina, Branchiomma bombyx, Fabricia stellaris, and of the Sabellariidae Sabellaria alveolata were studied in order to obtain detailed information on their chaetal arrangement and sites of chaetal origin. SEM investigations and computer-aided 3D-reconstructions provide deep insight into the spatial organization of the rami. Though differing externally, the principal chaetal arrangement and the location of the formative sites turned out to be almost identical within the species of Sabellidae. Most chaetae are aligned in straight transverse rows with a dorsal site of origin within neuropodia and a ventral one in notopodia as is common in sedentary polychaetes. Semicircular and spiral arrangements are revealed to be modified transverse rows. Only in thoracic notopodia does an additional dorsocaudal formative site form distinct rows. The chaetal inversion in Sabellidae is additionally characterized by an abrupt change of capillary chaetae and uncini along with a sudden change of the parapodial morphology at the border between thorax and abdomen. All chaetae of S. alveolata are aligned in transverse rows with the same location of the formative sites as in sabellids and other sedentary polychaetes. However, in contrast to sabellids the chaetae are not inverted across a parathoracic abdominal border. Moreover, there is no inversion of the parapodial structure from parathorax to abdomen and the neuropodial chaetal composition changes gradually from parathorax to abdomen. The chaetal arrangement in Sabellariidae thus cannot be described as inverted along the body-axis as in Sabellidae. Evolutionary steps implied by the assumption of an inverted chaetal pattern in a supposed common ancestor are discussed. It is concluded that the specific chaetal arrangement of Sabellidae and Sabellariidae arose independently and therefore provides no support for a sistergroup relationship of sabellids and sabellariids.     

Evidence for a role of Collapsin response mediator protein-2 in signaling pathways that regulate the proliferation of non-neuronal cells

Collapsin response mediator protein-2 or Crmp-2 plays a critical role in the establishment of neuronal polarity. In this study, we present evidence that apart from its functions in neurodevelopment, Crmp-2 is also involved in pathways that regulate the proliferation of non-neuronal cells through its phosphorylation by regulatory proteins. We show that Crmp-2 undergoes dynamic phosphorylation changes in response to contact inhibition-induced quiescence and that hyperphosphorylation of Crmp-2 occurs in a tumor. We further suggest that de-regulation of Crmp-2 phosphorylation levels at certain amino acid residues may lead to aberrant cell proliferation and consequently, tumorigenesis.     

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10^-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.     

The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus- Thermus group and cyanobacteria

The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks. These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon. The Hsp40 homolog from D. proteolyticus was found to be lacking a central 204 base pair region present in H. cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs. The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested. This unique deletion and the clustering of homologs from the Deinococcus–Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S. Gupta et al., J Bacteriol 179:345–357, 1997). Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences. Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited. However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences. Received: 30 January 1997 / Accepted: 22 March 1997     

Evidence for the close linkage between lymphocyte cytosol polypeptide with molecular weight of 64,000 (LCP1) and esterase D.

The genetic linkage of the loci for lymphocyte cytosol polypeptide with molecular weight of 64,000 (LCP1) and esterase (ESD) were examined by two-dimensional gel electrophoresis using blood from four informative families. No recombinants were observed in the four families totaling 17 children, giving a summed lod score of 4.221 at recombination fraction 0. This result indicates that the gene for LCP1 is closely linked to the ESD locus, which is assigned to the chromosomal region 13q14.11. This finding also suggests that the LCP1 locus is linked to the retinoblastoma gene, which is assigned to the same chromosomal region and closely linked to the ESD locus.