Expansion of Human and Murine Hematopoietic Stem and Progenitor Cells Ex Vivo without Genetic Modification Using MYC and Bcl-2 Fusion Proteins

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.     

Ultra-potent P1 modified arylsulfonamide HIV protease inhibitors: the discovery of GW0385

A novel series of P1 modified HIV protease inhibitors was synthesized and evaluated for in vitro antiviral activity against wild-type virus and protease inhibitor-resistant viruses. Optimization of the P1 moiety resulted in compounds with femtomolar enzyme activities and cellular antiviral activities in the low nanomolar range culminating in the identification of clinical candidate GW0385.     

Cuttlefish use visual cues to control three-dimensional skin papillae for camouflage

Cephalopods (octopus, squid and cuttlefish) are known for their camouflage. Cuttlefish Sepia officinalis use chromatophores and light reflectors for color change, and papillae to change three-dimensional physical skin texture. Papillae vary in size, shape and coloration; nine distinct sets of papillae are described here. The objective was to determine whether cuttlefish use visual or tactile cues to control papillae expression. Cuttlefish were placed on natural substrates to evoke the three major camouflage body patterns: Uniform/Stipple, Mottle and Disruptive. Three versions of each substrate were presented: the actual substrate, the actual substrate covered with glass (removes tactile information) and a laminated photograph of the substrate (removes tactile and three-dimensional information because depth-of-field information is unavailable). No differences in Small dorsal papillae or Major lateral mantle papillae expression were observed among the three versions of each substrate. Thus, visual (not tactile) cues drive the expression of papillae in S. officinalis. Two sets of papillae (Major lateral mantle papillae and Major lateral eye papillae) showed irregular responses; their control requires future investigation. Finally, more Small dorsal papillae were shown in Uniform/Stipple and Mottle patterns than in Disruptive patterns, which may provide clues regarding the visual mechanisms of background matching versus disruptive coloration.     

The effect of breed and housing system on dairy cow feeding and lying behaviour

In Ireland there is growing interest in managing dairy cows on out-wintering pads (OWPs) during the winter, as a low cost alternative to housing indoors. This study investigated feeding and lying behaviour of two breeds of dairy cow (Holstein-Friesian and Norwegian Red) at pasture (PAS) and in winter (WIN) confinement. Cows (n = 36) were managed as one herd while lactating at PAS, then dried off on entering WIN on 17 November 2005 and assigned to one of the three treatments using a randomised complete block design: (1) indoor matted cubicles [IC], (2) unsheltered OWP [UP] and (3) sheltered OWP [CP] (feed system = concrete feedface). Feeding behaviour was recorded for 1 × 24 h period during PAS and WIN using IGER grazing behaviour recorders. Standing/lying was recorded every 5 min for 2 × 24 h periods at PAS and 1 × 24 h period during WIN using modified voltage dataloggers (Tinytag Plus, Chichester, UK). Although not compared statistically, cows spent more time feeding at PAS (530.7 ± 69.66 min/day) than in any winter confinement system (UP, IC, CP = 453.9 ± 37.36, IC = 462.7 ± 37.31 and CP = 505.9 ± 37.36 min/day). In contrast, cows spent more time lying during the winter period (UP, IC, CP = 11.7 ± 0.45, IC = 10.8 ± 0.39 and CP = 11.0 ± 0.39 h/day) than when at PAS (9.4 h/day). Holstein-Friesian cows had a higher bite rate and fewer mastications while feeding, than Norwegian Red cows (P < 0.05 for both). Shorter feeding times at WIN were likely primarily due to lower metabolic requirements associated with the dry period, although the higher fibre content of the silage may also have contributed. The increase in ruminating times between PAS and WIN is likely also due to the higher fibre content of silage than grass. Differences in feeding behaviour between breeds may be indicative of reduced motivation to ingest food quickly. This may be an indirect consequence of a selection index that has led to improvements in body condition and self-maintenance. The similarity in lying and feeding times during WIN indicates that during these winter weather conditions the feeding and lying behaviour of cows on unsheltered OWPs was not inhibited compared with sheltered cows.     

A radiometric method for objectively screening large numbers of compounds against Pneumocystis carinii in vitro.

A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii cultured in vitro, and the subsequent development of a highly sensitive and reproducible 96-well microtitre plate drug screening system. Incorporation of [3H]pABA under optimized conditions has been utilized as a selective indicator of the in vitro viability of P. carinii against which the inhibitory effects of potential drugs were quantified. The anti-Pneumocystis agents pentamidine, sulfamethoxazole, 566C80 and piritrexim gave median inhibitory concentration values of 7.3, 0.1, 1.4 and approximately 100 microM, respectively in this assay. The results suggest that this 96-well plate P. carinii [3H]pABA-incorporation system is suitable as a rapid high throughput primary in vitro screen for detecting compounds with anti-Pneumocystis activity.     

Linking number anomalies in DNA under conditions close to condensation

Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fatal penetrating skin ulcers in laboratory-reared octopuses

Young Octopus joubini and O. briareus (35 to 60 days old) that were being reared in high-density groups for biomedical studies developed skin ulcers, whereas octopuses reared in individual containers in the same culture system were disease-free. The ulcers first affected the epidermis of the mantle, and then penetrated downward through the dermis and underlying muscle tissue. Four stages of ulceration were observed. Untreated octopuses with ulcers always died, usually within 4 days. Five species of bacteria were isolated from ulcers: Vibrio alginolyticus, V. damsela, Pseudomonas stutzeri, and Aeromonas caviae from O. joubini; and V. parahaemolyticus, V. damsela, and P. stutzeri from O. briareus. Bacteria were found during all stages of the ulceration. Healthy O. joubini were infected experimentally with four species of bacteria, and V. alginolyticus produced skin ulcers within 2 days. The ulceration was treated with nifurpirinol, and complete healing of the skin occurred within 2 months. The ulcers were probably species-specific because O. maya and O. bimaculoides that were reared in the same culture systems were not affected. The cause of the ulceration was probably an increase in contact among crowded octopuses that produced skin abrasions which were invaded by opportunistic bacteria.     

Comparison of the linear least mean squares procedure and the vector projection method of spectral analysis.

A mathematical analysis of two methods commonly used for the determination of fractions of secondary structure from circular dichroism spectral data—the vector projection method of Baker &; Isenberg [Biochem.15, 629 (1976)] and the classical linear least mean squares procedure-reveals that the two procedures handle errors of all types in an equivalent fashion and yield identical values of the fractional amounts of secondary structure present, as long as the same method of integration of the data is employed. Analyses of simulated data show that the method of integration [rectangular, trapezoidal or parabolic (Simpson's Rule)] has an insignificant effect on the calculated values of the fractions of secondary structure present. Our study also shows that the insensitivity of the fraction of α helix calculated using a given random coil reference spectrum is a natural consequence of certain intrinsic properties of the reference spectra of the α helix, β structures and random coil forms, rather than the method of calculation employed.