Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers

Cell-free translation of beta-tubulin mRNA generates full length beta-tubulin polypeptides distributed in three molecular forms: a high molecular weight lysate-associated form, the free beta-tubulin subunit, and the alpha beta-heterodimer (Yaffe, M.B., Farr, G. W., and Sternlicht, H. (1988) J. Biol. Chem. 263, 16023-16031). A quantitative assay system for these three forms was developed and used to measure the rates of incorporation/exchange of the newly synthesized free beta-subunit and the high molecular weight form into tubulin heterodimers following incubation of the 35S-translation products with unlabeled bovine tubulin dimer. This exchange process was found to be slow and strongly temperature-dependent. The half-lives for exchange ranged from 12.5 min at 37 degrees C to 17.5 h at 0 degree C with a measured activation energy of 22.5 kcal/mol. Microtubule-associated proteins appeared to play no role in the exchange process, since identical exchange rates were observed regardless of whether microtubule protein or phosphocellulose-purified tubulin was used as the source of tubulin dimer. Surprisingly, the exchange rates were found to be independent of dimer concentration. We interpret these results as evidence for a rate-limiting, slow conformational change that occurs within the newly synthesized beta-subunits prior to their association with alpha-tubulin to generate the alpha beta-hetero-dimer.     

Orientation of dioxygen bound to cobalt(II) bleomycin-DNA fibers

The molecular oxygen adduct of Co(II)-bleomycin is stable for long periods when bound to salmon sperm DNA at large ratios of polymer to drug. According to ESR studies of orientation of the paramagnetic complex associated with DNA fibers, the oxygen-oxygen bond is restricted to a plane perpendicular to the fiber axis. Thus, one can define three g values for the adduct 2.104, 2.016, and 2.000, one parallel to the fiber axis and two orthogonal to it. There is no change in orientation over the range of 77 K to ambient temperature. Furthermore, there is no difference in results at a series of relative humidities ranging from less than 76% where bulk DNA alone assumes an A conformation to 95% where it is primarily B DNA. A structural model is presented for the geometry of the metal binding domain of O2-Co-bleomycin in relationship to the fiber axis of DNA.     

Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.     

Problems in polychaete systematics

Some of the intriguing issues in current polychaete systematics are reviewed. (1) The root of the `polychaete' tree. Currently there are two major hypotheses concerning the root position among polychaetes. One is based on rooting cladograms with outgroups such as Mollusca and result in simple-bodied taxa such as Opheliidae and Questidae forming a basal annelid grade along with Clitellata. Other hypotheses do not use outgroup rooting but involve scenarios on the evolution of the group and would place taxa in Aciculata as basal annelids, thus making Aciculata and Phyllodocida paraphyletic. Molecular sequence data has been of little help in resolving this issue thus far, largely due to limited taxon sampling. (2) Paraphyly. Owing, in part, to a tradition involving the emphasis on differences among taxa, and the application of Linnean ranks (e.g., family), paraphyly is undoubtedly a widespread phenomenon in polychaete systematics. An example of this has been proposed already for Spionidae. If the tree topology and rooting used by Blake & Arnofsky (1999) is correct, Spionidae is made paraphyletic by the recognition of the following four family-ranked taxa; Trochochaetidae, Poecilochaetidae, Longosomatidae and Uncispionidae. Another possible example is seen with Cirratulidae. A preliminary cladistic analysis shows that it is entirely possible that seven other taxa recognised as families may be nested within Cirratulidae. These include Acrocirridae, Ctenodrilidae, Fauveliopsidae, Flabelligeridae, Flotidae, Poeobiidae and Sternaspidae. (3) Problematic taxa. Apart from the problems exposed by the analysis of Cirratuliformia, the position of some of these groups, such as Aberranta, Alciopidae, Hesionides,Lopadorhynchidae, Microphthalmus,Nerillidae, Spinther,Tomopteridae and Sabellariidae, is discussed.     

Relationship of Aerial Broad Band Reflectance to Meloidogyne incognita Density in Cotton

Aerial images were obtained on 22 July 1999 and 4 August 2000 from five cotton sites infested with Meloidogyne incognita. Images contained three broad bands representing the green (500-600 nm), red (600-700 nm), and near-infrared (700-900 nm) spectrum. Soil samples were collected and assayed for nematodes in the fall at these sites. Sampling locations were identified from images, by locating the coordinates of a wide range of light intensity (measured as a digital number) for each single band, and combinations of bands. There was no single band or band combination in which reflectance consistently predicted M. incognita density. In all 10 site-year combinations, the minimum number of samples necessary to estimate M. incognita density within 25% of the population mean was greater when sampling by reflectance-based classes (3 to 4 per site) than sampling based on the entire site as one unit. Two sites were sampled at multiple times during the growing season. At these sites, there was no single time during the growing season optimal to take images for nematode sampling. Aerial infrared photography conducted during the growing season could not be used to accurately determine fall population densities of M. incognita.     

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

An ATP-Sensitive K+ Current that Regulates Progression Through Early G1 Phase of the Cell Cycle in MCF-7 Human Breast Cancer Cells

Whole-cell recordings were used to identify in MCF-7 human breast cancer cells the ion current(s) required for progression through G1 phase of the cell cycle. Macroscopic current-voltage curves were fitted by the sum of three currents, including linear hyperpolarized, linear depolarized and outwardly rectifying currents. Both linear currents, but not the outwardly rectifying current, were increased by 1 μm intracellular Ca²⁺ and blocked by 2 mm intracellular ATP. When tested at concentrations previously shown to inhibit proliferation by 50%, linogliride, glibenclamide and quinidine inhibited the linear hyperpolarized current, and quinidine and linogliride inhibited the linear depolarized current; none of these agents affected the outwardly rectifying current. In contrast, tetraethylammonium completely inhibited the outwardly rectifying current, but did not inhibit either linear current. Changing the bath solution to symmetric K⁺ shifted the reversal potential of the linear hyperpolarized current from near the K⁺ equilibrium potential (−84 mV) to −4 mV. Arrest of the cell cycle in early G1 by quinidine was associated with significantly smaller linear hyperpolarized currents, without a change in the linear depolarized or outwardly rectifying currents, but this reduction was not observed with arrest by lovastatin at a site ≈6 hr later in G1. The linear hyperpolarized current was significantly larger in ras-transformed than in untransformed cells. We conclude that the linear hyperpolarized current is an ATP-sensitive K⁺ current required for progression of MCF-7 cells through G1 phase. Received: 22 January 1999/Revised: 11 May 1999     

CD44-deficient mice develop normally with changes in subpopulations and recirculation of lymphocyte subsets.

Cell adhesion molecules are considered to be pivotal elements required for proper embryo development. The transmembrane glycoprotein CD44, which is expressed in numerous splice variants on the surface of many different cell types and tissues, has been suggested to be involved in several physiological processes such as cell-cell interactions, signal transduction, and lymphocyte homing and trafficking during embryogenesis and in the adult organism. Some splice variants are thought to play an important role in tumor progression. To investigate the physiological roles of CD44 in vivo, we abolished expression of all isoforms of CD44 in mice by targeted insertion of a lacZ/neo cassette into the reading frame of the leader peptide. CD44-deficient mice are viable without obvious developmental defects and show no overt abnormalities as adults. However, CD44-deficient lymphocytes exhibit impaired entry into the adult thymus, although lymphocyte development is apparently unaltered. Our data indicate that all splice variants of CD44 are dispensable for embryonic development and implicate a critical function for CD44 in lymphocyte recirculation.