Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved

Role of the α-Amino Groups of the α and β Chains of Human Hemoglobin in Oxygen-linked Binding of Carbon Dioxide

Abstract Human hemoglobin has been reacted with potassium cyanate and purified to yield three species, α2cβ2c,α2cβ2, and α2β2c, where superscript c denotes specific reaction of cyanate with the α-amino group of the particular chain. The effect of carbon dioxide on the oxygen affinity of these species in the presence and in the absence of 2,3-diphosphoglycerate has been measured. Carbon dioxide has no effect on the oxygen affinity of α2cβ2c, confirming that the usual lowering of the oxygen affinity of carbon dioxide in normal hemoglobin is mediated by the α-amino groups of the α and β chains. The lowering of the oxygen affinity of α2β2c by carbon dioxide was not affected by the presence or absence of 2,3-diphosphoglycerate, showing that 2,3-diphosphoglycerate does not interfere with the oxygen-linked binding of carbon dioxide at the α chain α-amino group. In α2cβ2 there was a much larger effect of carbon dioxide on the oxygen affinity in the absence of 2,3-diphosphoglycerate than in α2β2c; however, on addition of 2,3-diphosphoglycerate the effect of carbon dioxide on the oxygen affinity of α2cβ2 was much smaller and similar to that occurring in α2β2c. This shows that there is a large difference in the carbon dioxide binding constants of the β chain α-amino group in the oxy and deoxy forms of human hemoglobin, and that 2,3-diphosphoglycerate suppresses this difference, probably by binding strongly to the β chain α-amino group of deoxyhemoglobin and displacing any bound carbon dioxide

hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends

Glycaemic Control among Rural Health Consumers: A Retrospective Study of a Diabetes Center

Abstract Aim: The aim of this paper is to highlight the successes of and challenges faced by a publically funded diabetes center in a regional area. Methods: Demographic and laboratory cross sectional data were collected from electronic patient records. Data from a patient's very first test undertaken when attending the hospital and the latest test undertaken at the Diabetes center were noted and included age, sex, residential postcode and glycated haemoglobin (HbA1c) levels. Results: A third of patients reached the therapeutic guideline of 'very good control' for HbA1c levels. Females had lower Hb1Ac levels, while males and those that lived further away from the diabetes center had higher levels of HbA1c. However, a significant improvement in glycaemic control among men and those who lived 'out of town' was noted, while the corresponding pattern for women was not evident. Conclusion: The study demonstrated that there was an overall improvement in diabetes control among health consumers who attend the regional diabetes center, however, female patients residing in town showed a negligible change over time. At risk' patient groups may need further targeting for intensive intervention to achieve optimal diabetes control, even within the diabetes center

Structural role of Sfi1p–centrin filaments in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–centrin complexes containing several repeats show Sfi1p as an α helix with centrins wrapped around each repeat and similar centrin–centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly

Antioxidant activity, total phenolics and flavonoids contents: should we ban in vitro screening methods?

As many studies are exploring the association between ingestion of bioactive compounds and decreased risk of non-communicable diseases, the scientific community continues to show considerable interest in these compounds. In addition, as many non-nutrients with putative health benefits are reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators, measurement of antioxidant activity using in vitro assays has become very popular over recent decades. Measuring concentrations of total phenolics, flavonoids, and other compound (sub)classes using UV/Vis spectrophotometry offers a rapid chemical index, but chromatographic techniques are necessary to establish structure-activity. For bioactive purposes, in vivo models are required or, at the very least, methods that employ distinct mechanisms of action (i.e., single electron transfer, transition metal chelating ability, and hydrogen atom transfer). In this regard, better understanding and application of in vitro screening methods should help design of future research studies on ‘bioactive compounds’

Architecture of the Human Ndc80-Hec1 Complex, a Critical Constituent of the Outer Kinetochore

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex