The role of hair follicle nestin‐expressing stem cells during whisker sensory‐nerve growth in long‐term 3D culture

We have previously reported that nestin‐expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND‐GFP mice), and were placed in 3D histoculture supported by Gelfoam®. β‐III tubulin‐positive fibers, consisting of ND‐GFP‐expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F‐actin. These findings indicate that β‐III tubulin‐positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND‐GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin‐expressing stem cells in the hair follicle is for growth of the follicle sensory nerve. J. Cell. Biochem. 114: 1674–1684, 2013. © 2013 Wiley Periodicals, Inc.     

Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor-scaffold collagen in the primary and metastatic tumor microenvironment

Human osteosarcoma 143B cells were previously stably transfected with an α_v integrin green flourescent protein (GFP) vector. 143B cells expressing α_v integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of α_v integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing α_v integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing α_v integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing α_v integrin-GFP in the lung. The current study demonstrates the importance of α_v integrin interaction with stromal elements in osteosarcoma.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Optimal foraging: food patch depletion by ruddy ducks

Summary I studied the foraging behavior of ruddy ducks (Oxyura jamaicensis) feeding on patchily distributed prey in a large (5-m long, 2-m wide, and up to 2-m deep) aquarium. The substrate consisted of a 4x4 array of wooden trays (1.0-m long, 0.5-m wide, and 0.1-m deep) which contained 6 cm of sand. Any tray could be removed from the aquarium and loaded with a known number of prey. One bird foraged in the aquarium at a time; thus, by removing a food tray after a trial ended and counting the remaining prey, I calculated the number of prey consumed by the bird. I designed several experiments to determine if ruddy ducks abandoned a food patch in a manner consistent with the predictions of a simple, deterministic, patch depletion model. This model is based on the premise that a predator should maximize its rate of net energy intake while foraging. To accomplish this, a predator should only remain in a food patch as long as its rate of energy intake from that patch exceeds the average rate of intake from the environment. In the majority of comparisons, the number of food items consumed by the ruddy ducks in these experiments was consistent with the predictions of the foraging model. When the birds did not forage as predicted by the model, they stayed in the patch longer and consumed more prey than predicted by the model. An examination of the relation between rate of net energy intake and time spent foraging in the food patch indicated that by staying in a patch longer than predicted, the ruddy ducks experienced only a small deviation from maximum rate of net energy intake. These results provided quantitative support for the prediction that ruddy ducks maximize their rate of net energy intake while foraging.     

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC₅₀ values below 1 μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC₅₀ values lying in a range of 11–45 nM. These most potent compounds might be bi-substrate analogues.     

Overexpression of catalase or Bcl-2 delays or prevents alterations in phospholipid metabolism during glucocorticoid-induced apoptosis in WEHI7.2 cells

Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2.     

Identification of P‐glycoprotein co‐fractionating proteins and specific binding partners in rat brain microvessels

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.

     

Effects of balance training using wobble boards in the elderly

Few studies have examined balance training of elderly people using wobble boards. This study assessed the effects of wobble board balance training on physical function in institutionalized elderly people. This study examined 23 subjects (age 84.2 ± 5.9 years) who lived in a nursing home. The exercise program for the training group comprised balance training standing on a wobble board for 9 weeks, twice a week. In all, 11 training group subjects and 11 control group subjects completed this study. After 9 weeks, standing time on a wobble board, standing time on a balance mat, and maximum displacement distance of anterior-posterior center of pressure in the training group were significantly greater than those of the control group. Frequency analysis revealed that the power spectrum in 0.1-0.2 Hz significantly increased in the training group. These results suggest that wobble board training is effective for elderly people to improve their standing balance, by which they frequently control their center of gravity and maintain a standing posture on unstable surface conditions.     

Loss of Intracellular Putrescine Pool-Size Regulation Induces Apoptosis

Synthesis and uptake are two important regulated mechanisms by which eukaryotic cells maintain polyamine levels. The role that loss of synthesis and/or uptake regulation plays in mediating putrescine toxicity was investigated by comparing toxicity in an ornithine decarboxylase (ODC)-deficient Chinese hamster ovary cell line (C55.7) with a functional putrescine transport system and an ODC-overproducing rat hepatoma cell line (DH23b), which are transport regulation deficient. When C55.7 cells were transfected with either mouse ODC (M) or trypanosome ODC (Tb), intracellular putrescine content increased slightly in C55.7(Tb-ODC), compared to C55.7(M-ODC), due to the lack of response of Tb-ODC to polyamine regulation. The increase in putrescine content resulting from loss of ODC regulation had no impact on cell growth and viability. When the feedback repression of polyamine uptake was blocked with cycloheximide, C55.7 cells transfected with either ODC construct accumulated very high levels of putrescine from the medium, and underwent apoptosis in a putrescine dose-dependent manner. A similar correlation of deregulated putrescine uptake and increased apoptotic cells was observed in DH23b cells. These data demonstrate that loss of feedback regulation on the polyamine transport system, but not ODC activity, is sufficient to induce apoptosis. Thus, downregulation of the transport system is necessary to prevent accumulation of cytotoxic putrescine levels in rodent cells.