Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer or elk PrP in transgenic mice has induced susceptibility to chronic wasting disease (CWD), the prion disease of cervids. In the current experiments, transgenic mice expressing two naturally occurring allelic variants of deer PrP with either glycine (G) or serine (S) at residue 96 were found to differ in susceptibility to CWD infection. G96 mice were highly susceptible to infection, and disease appeared starting as early as 160 days postinfection. In contrast, S96 mice showed no evidence of disease or generation of disease-associated protease-resistant PrP (PrPres) over a 600-day period. At the time of clinical disease, G96 mice showed typical vacuolar pathology and deposition of PrPres in many brain regions, and in some individuals, extensive neuronal loss and apoptosis were noted in the hippocampus and cerebellum. Extraneural accumulation of PrPres was also noted in spleen and intestinal tissue of clinically ill G96 mice. These results demonstrate the importance of deer PrP polymorphisms in susceptibility to CWD infection. Furthermore, this deer PrP transgenic model is the first to demonstrate extraneural accumulation of PrPres in spleen and intestinal tissue and thus may prove useful in studies of CWD pathogenesis and transmission by oral or other natural routes of infection.     

Application of cationic conjugated polymers in microarrays using label-free DNA targets

A fluorescence-based microarray technique that does not require target DNA labeling is detailed. This 'label-free' approach utilizes a cationic, water-soluble conjugated polymer PFBT (poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide]), and neutral PNA (peptide nucleic acid) hybridization probes. DNA hybridization to immobilized PNA spots results in a change in the net charge at that particular surface. Electrostatic interactions between the cationic polymer and negatively charged DNA bind the polymer to the hybrid DNA/PNA complex. By exciting the conjugated polymer at 488 nm on a commercial microarray scanner, the presence of the target is directly indicated by the fluorescence emission of the polymer. This feature eliminates the necessity of target labeling required in traditional microarray protocols. There are five steps involved in the procedure before scanning or imaging the array: (i) slide hydration, (ii) target hybridization, (iii) post-hybridization washing, (iv) polymer application and (v) polymer washing. Each step takes 20 min to 1 h. The overall protocol requires approximately 2-3 h.     

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2FGC transporter through a rigid-body mechanism to extract LPS

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB₂FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.     

Subject-specific,whole-body models of the stooped posture with a personal weight transfer device

A prior laboratory study found that when wearing a weight transfer device in the stooped posture, trunk flexions were reduced, and subjects who did not experience flexion–relaxation of the erector spinae had reduced back muscle activity. Whole-body musculoskeletal models, which included individual passive torso stiffness and anthropometry, were implemented to predict loads in the passive tissues of the back and the leg joints. Results predicted that when wearing the device in the stooped posture, compression and shear forces at the L5–S1 level were reduced by 13% and 12% respectively. Internal loads in the leg joints were reduced between 10% and 31%. Much of the reduction in joint loads may be a result of the device’s ability to limit torso flexion during stoop, rather than a transferring of load. While these results show possible benefit in the short-term, further study is needed on the long-term effects to determine if the device is an effective intervention for those who use the stooped posture routinely.     

Transmembrane potential responses during HL-60 promyelocyte differentiation

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC₂-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by K_Ca channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl⁻ conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions. © 1996 Wiley-Liss, Inc.     

Effect of gestational sex steroid exposure on limb development and endochondral ossification in the pregnant C57Bl/6J mouse: I. Medroxyprogesterone acetate

Although data supporting the teratogenic potential of intrauterine progestin exposure is lacking, concern persists among some individuals within the scientific community that these drugs have the potential for nongenital teratogenesis, especially with regard to limb reduction defects. Our laboratory has been interested in the ontogeny of steroid receptors in the developing embryo and in the role of steroid-receptor interactions in limb development, particularly the process of endochondral ossification. Since limb reduction defects can be produced from abnormal processes that are operative during organogenesis or during midgestation (vascular disruption) we have designed an animal study whereby embryos were exposed to sex steroids throughout organogenesis and fetal development. The present study assesses the effects of medroxyprogesterone acetate (MPA) on intrauterine endochrondral bone development specifically, as well as overall embryo-fetal development. Primagravid C57Bl/6J mice were treated via subdermal pellets which deliver MPA at dosages of 5.0, 50.0, and 500.0 mg/kg/day on gestational days 7 through 19. These doses were 25-, 250-, and 2,500-fold higher on a mg/kg basis than the human dose equivalent (HDE). No increases in nongenital malformations were noted at any evaluated MPA dosage level. At 25 X the HDE, MPA did not influence endochondral bone development as evidenced by a lack of significant effects on assessed bone growth parameters. In the 250- and 2,500-fold HDE dosage groups, MPA was shown to exert an embryotoxic effect inducing 48 and 100% resorptions respectively. Mean embryo weights/litter were significantly reduced by MPA exposure at 250 X the HDE. Intrauterine exposure to 250 X the MPA HDE induced reductions in humeral and femoral diaphyseal length in proportion to a reduction in overall growth. The data demonstrate that MPA, administered at dosages of up to several orders of magnitude in excess of the HDE and which permitted embryo survival, did not induce increases in the frequency of nongenital teratogenesis at any dose or gestational stage. Importantly, limb reduction defects were not noted even in instances where the dosage of MPA induced an inhibition of endochondral bone growth.     

Mechanisms underlying insect freeze tolerance

Freeze tolerance – the ability to survive internal ice formation – has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze‐tolerant insects accumulate many potentially protective molecules, there is no apparent ‘magic bullet’ – a molecule or class of molecules that appears to be necessary or sufficient to support this cold‐tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze‐tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze‐tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post‐thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold‐ and desiccation‐tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold‐tolerance strategy that has evolved multiple times, we suggest that a process‐focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis‐driven research to understand better how insects survive internal ice formation.     

Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.     

Mutations in the S6 Gate Isolate a Late Step in the Activation Pathway and Reduce 4-AP Sensitivity in Shaker Kv Channel

K_v channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.