Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7

CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.     

O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.     

Regional registration of [6‐14C]glucose metabolism during brain activation of α‐syntrophin knockout mice

α‐Syntrophin is a component of the dystrophin scaffold‐protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α‐Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K⁺ homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4‐mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [¹⁴C]metabolites during sensory stimulation. Conscious KO and wild‐type mice were pulse‐labeled with [6‐¹⁴C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer‐assisted brain imaging or analysis of [¹⁴C]metabolites in extracts, respectively. High‐resolution autoradiographic assays detected a 17% side‐to‐side difference (p < 0.05) in inferior colliculus of KO mice, not wild‐type mice. However, there were no labeling differences between KO and wild‐type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4‐mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Progress in understanding the biosynthesis of amylose

The storage of glucose in insoluble granules is a distinctive feature of plant cells. Biosynthesis of amylose, the minor low molecular mass fraction of starch occurs from ADP-glucose. This takes place within the polysaccharide matrix through the action of granule-bound starch synthase, the major protein associated with the granule. Recently, amylose has been successfully synthesized in vitro from purified granules. Two models have been proposed to explain the mechanism of amylose synthesis in plants. The first calls for priming of synthesis through small-size malto-oligosaccharides. The second suggests that glucans are extended by granule-bound starch synthase from a high molecular mass primer present within the granule. This extension is terminated through cleavage to produce amylose. This process is subsequently repeated to give several rounds of amylose synthesis.     

Homogeneous and heterogeneous tertiary structure ensembles of amyloid-β peptides

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimer's disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.     

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein.

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.     

Sex differences in the response to environmental cues regulating seasonal reproduction in birds

Although it is axiomatic that males and females differ in relation to many aspects of reproduction related to physiology, morphology and behaviour, relatively little is known about possible sex differences in the response to cues from the environment that control the timing of seasonal breeding. This review concerns the environmental regulation of seasonal reproduction in birds and how this process might differ between males and females. From an evolutionary perspective, the sexes can be expected to differ in the cues they use to time reproduction. Female reproductive fitness typically varies more as a function of fecundity selection, while male reproductive fitness varies more as a function sexual selection. Consequently, variation in the precision of the timing of egg laying is likely to have more serious fitness consequences for females than for males, while variation in the timing of recrudescence of the male testes and accompanying territory establishment and courtship are likely to have more serious fitness consequences for males. From the proximate perspective, sex differences in the control of reproduction could be regulated via the response to photoperiod or in the relative importance and action of supplementary factors (such as temperature, food supply, nesting sites and behavioural interactions) that adjust the timing of reproduction so that it is in step with local conditions. For example, there is clear evidence in several temperate zone avian species that females require both supplementary factors and long photoperiods in order for follicles to develop, while males can attain full gonadal size based on photoperiodic stimulation alone. The neuroendocrine basis of these sex differences is not well understood, though there are many candidate mechanisms in the brain as well as throughout the entire hypothalamo-pituitary-gonadal axis that might be important.