Elastase in the different primary granules of the human neutrophil

Elastase in the human neutrophil is associated with various subpopulations of primary granules of different density. The proportion of this enzyme that is extracted with acetate pH 4.2 and cetyltrimethylammonium bromide varies in the different subpopulations. Nevertheless, the electrophoretic mobility and relative proportions of elastase isoenzymes is the same in both extracts from the different subpopulations. On stimulation of neutrophils with N-formylmethionylleucylphenylalanine, elastase is not released from the least dense subpopulation, whereas other two subpopulations do undergo degranulation to approximately the same extent. However, the release of elastase from these two denser granules differs after they are isolated and treated with calcium.     

Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction.

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, K(v)1.3, and [(3)H]dihydrocorreolide (diTC) binds with high affinity (K(d) approximately 10 nM) to membranes from Chinese hamster ovary cells that express K(v)1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F. , Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, K(v)3.2. Transferring the pore from K(v)1.3 to K(v)3.2 produces a chimera that binds peptidyl inhibitors of K(v)1.3 with high affinity, but not diTC. Transfer of the S(5) region of K(v)1.3 to K(v)3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with K(v)1.3, whereas transfer of the entire S(5)-S(6) domain results in a normal K(v)1.3 phenotype. Substitutions in S(5)-S(6) of K(v)1.3 with nonconserved residues from K(v)3.2 has identified two positions in S(5) and one in S(6) that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to K(v)3.2 after substitution of these three residues with the corresponding amino acids found in K(v)1.3. These results suggest that lack of sensitivity of K(v)3.2 to diTC is a consequence of the presence of Phe(382) and Ile(387) in S(5), and Met(458) in S(6). Inspection of K(v)1.1-1.6 channels indicates that they all possess identical S(5) and S(6) domains. As expected, diTC binds with high affinity (K(d) values 7-21 nM) to each of these homotetrameric channels. However, the kinetics of binding are fastest with K(v)1.3 and K(v)1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify K(v)1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.     

Exploring the Potential of Laser Ablation Carbon Isotope Analysis for Examining Ecology during the Ontogeny of Middle Pleistocene Hominins from Sima de los Huesos (Northern Spain)

Laser ablation of tooth enamel was used to analyze stable carbon isotope compositions of teeth of hominins, red deer, and bears from middle Pleistocene sites in the Sierra de Atapuerca in northern Spain, to investigate the possibility that this technique could be used as an additional tool to identify periods of physiological change that are not detectable as changes in tooth morphology. Most of the specimens were found to have minimal intra-tooth variation in carbon isotopes (< 2.3‰), suggesting isotopically uniform diets through time and revealing no obvious periods of physiological change. However, one of the two sampled hominin teeth displayed a temporal carbon isotope shift (3.2‰) that was significantly greater than observed for co-occurring specimens. The δ¹³C value of this individual averaged about -16‰ early in life, and -13‰ later in life. This isotopic change occurred on the canine crown about 4.2 mm from the root, which corresponds to an approximate age of two to four years old in modern humans. Our dataset is perforce small owing to the precious nature of hominid teeth, but it demonstrates the potential utility of the intra-tooth isotope profile method for extracting ontogenetic histories of human ancestors.     

Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.     

Association of LHIα(B870) Polypeptide with Phospholipids During Insertion in the Photosynthetic Membrane of an LHII− Mutant of Rhodobacter capsulatus

Membranes from in vivo labeled cells of Rhodobacter capsulatus U43[pTX35] grown photosynthetically carried 60% of the [³²P]-Pi in the “heavy” fraction (HM) after sucrose gradient sedimentation. Metal-chelating chromatography of either “heavy” or “light” (LM) membrane fractions rendered similar Bchl-protein complex profiles after octyl-glucoside treatment, including most of the radioactivity in the same corresponding elution fraction (F II). Similar labeling distribution of pigment-protein complexes was obtained for membranes of dark-grown cells induced by lowering oxygen tension. Fractions derived from HM showed highly labeled LHIα, whereas the same complex from LM was essentially [³²P]-Pi-free, as revealed by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a similar pattern for membranes isolated from cells photosynthetically or semiaerobically grown, being the most abundant: phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the phospholipids from HM comigrated with LHIα during SDS-PAGE and dissociated from the complexes only after solvent extraction and hydrophobic chromatography. However, a small amount remained always attached to LHIα, indicating an unusual strong interaction. These results suggest the existence of two operationally defined membrane regions carrying LHIα complexes differing in phosphorylation status and protein-phospholipid interaction. Received: 10 August 1996 / Accepted: 10 September 1996