Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Monokaryotic chloroplast mutation has no effect on non-Mendelian transmission of chloroplast and mitochondrial DNA in Chlamydomonas species

Summary. We studied whether the monokaryotic chloroplast (moc) mutation affects the transmission of chloroplast and mitochondrial DNA in Chlamydomonas species. We used a previously isolated moc mutant from our cell line G33, which had only one large chloroplast nucleus. To obtain zygotes we crossed the mutant cells with wild-type cells, and mutant cells with receptive mates (females [mt+] with males [mt–]). In these zygotes, we recorded preferential dissolution of mt– parental chloroplast nuclei and fusion of the two cell nuclei. Antibiotic-resistance markers of chloroplast DNA were maternally transmitted in all crosses. PCR analysis of the cytochrome b (cob) gene sequence showed that the mitochondrial DNA was paternally transmitted to offspring. These results suggest that the moc mutation did not affect the organelle DNA transmission.Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan.     

Roles of oxytocin in spatial learning and memory in the nucleus basalis of Meynert in rats

The present study was performed to explore the role of oxytocin (OT) in spatial learning and memory in the nucleus basalis of Meynert (NBM) of rats. The latency, distance and swimming path to find the platform were tested by Morris water maze and recorded by a video camera connected to a computer. Intra-NBM injections of 2 or 10 nmol of OT, but not 0.2 nmol of OT, induced significant increase on the latency of spatial learning. Rats receiving intra-NBM administrations of 2 or 10 nmol of OT showed a more random search pattern. There were no significant changes in the swimming speed in Morris water maze test after the injection of OT. Furthermore, the impaired effect of OT on the latency of spatial learning was blocked by intra-NBM injection of the selective OT antagonist Atosiban, indicating that the effect of OT was mediated by OT receptor in the NBM of rats. Moreover, there were no influences of OT or Atosiban on the retention performance in rats. The results suggest that OT plays an inhibitory role in spatial learning in the NBM; the effect is mediated by OT receptor.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.     

Nuclear crystals, lampbrush-chromosome-like structures, and perinuclear cytoskeletal elements associated with nuclear fragmentation in characean internodal cells

Summary This article gives a survey of nucleus-associated structures and inclusions in a diverse range of characean algae includingChara braunii Gm.,Chara corallina Klein ex Willd., em. R.D.W.,Nitella cristata A.Br., em. R.D.W.,Nitella flexilis (L.) Ag.,Nitella furcata (Roxb. ex Bruz.) Ag. em. R.D.W.,Nitella hyalina (DC.) Ag.,Nitella pseudoflabellata A.Br., em. R.D.W.,Nitella pseudoflabellata var.imperialis T.F.A.,Nitella translucens var.axillaris (A.Br.) R.D.W. andNitellopsis obtusa (Desv. in Lois.) J.Gr. Lampbrushchromosome-like structures were found in nuclei ofNitella flexilis andNitellopsis obtusa and seem to be involved in the distribution of genetic material during nuclear fragmentation. Intranuclear tubular crystals of unknown protein composition were present in all species, especially in young, elongating cells, and could be important for establishing the main axis of the nuclei. Spindle-shaped protein crystals that originate in the nucleus and are released into the cytoplasm upon nuclear degeneration were observed in branchlet internodal cells of one population ofNitella flexilis. Perinuclear microtubules were present in all species, but perinuclear actin fibrils were hitherto only found in mostNitella species and inNitellopsis obtusa. None of these nucleus-associated structures seems to be responsible for the formation of constrictions leading to nuclear fragmentation. These constrictions were perpendicular to the main axis of the nucleus and symmetrical in theNitella species but asymmetric inC. braunii, C. corallina, and inNitellopsis obtusa. Statistical analysis of nuclear size, number and constriction sites indicate that fragmentation is a nonsynchronous process independent of the light-dark cycle.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - LCLS lampbrush chromosome-like structure(s) Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.