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.     

Amelioration of cognitive deficits in plaque‐bearing Alzheimer's disease model mice through selective reduction of nascent soluble A42 without affecting other A pools

Given that amyloid‐β 42 (Aβ42) is believed to be a culprit in Alzheimer's disease (AD), reducing Aβ42 production should be a potential therapeutic approach. γ‐Secretase modulators (GSMs) cause selective reduction of Aβ42 or both reduction of Aβ42 and Aβ40 without affecting total Aβ through shifting the γ‐cleavage position in amyloid precursor protein. We recently reported on GSM‐2, one of the second‐generation GSMs, that selectively reduced brain Aβ42 level and significantly ameliorated cognitive deficits in plaque‐free 5.5‐month‐old Tg2576 AD model mice. Here, we investigated the effects of GSM‐2 on 10‐, 14‐, and 18‐month‐old mice which had age‐dependent increase in amyloid plaques. Eight‐day treatment with GSM‐2 significantly ameliorated cognitive deficits measured by Y‐maze task in the mice of any age. However, GSM‐2 reduced brain soluble Aβ42 only in 10‐month‐old mice. In contrast, GSM‐2 markedly reduced newly synthesized soluble Aβ42 in both 10‐ and 18‐month‐old mice with similar efficacy when measured using the stable isotope‐labeling technique, suggesting that nascent Aβ42 plays a more significant role than plaque‐associated soluble Aβ42 in the cognitive deterioration of Tg2576 mice. These findings further indicate the potential utility of approach to reducing Aβ42 synthesis in AD therapeutic regimens.     

Ly6C+Ly6G− Myeloid‐derived suppressor cells play a critical role in the resolution of acute inflammation and the subsequent tissue repair process after spinal cord injury

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C⁺Ly6G^? immature monocyte fraction exhibited the same characteristics as myeloid‐derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C⁺Ly6G^? fraction prior to injury by anti‐Gr‐1 antibody (clone: RB6‐8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo‐generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C⁺Ly6G^? fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC‐based strategy that can be applied to acute inflammatory diseases.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Responses of Isolated Plant Mitochondria to Photosynthesis Inhibiting Acylanilides

During experiments to elucidate the mode of action of photosynthesis inhibiting acylanilide type herbicides, the effects of various acylanilides on respiration and oxidative phosphorylation of isolated plant mitochondria were studied. The results showed that some acylanilides acted as uncouplers of oxidative phosphorylation: 1) Some stimulated the ADP-limited state 4 respiration of isolated mitochondria depriving them of their respiratory control ability during succinate oxidation. 2) Those which stimulated state 4 respiration interfered with oxidative phosphorylation to degenerate the P/O ratio.

The following relationships between chemical structure of acylanilides and their biological activities were demonstrated: 3) Among various ring-chlorinated propionanilides, the activity of 3′,4′-DCPA was especially prominent. 4) Almost all the side chain-substituted 3′,4′-dichloroacylanilides tested were effective. 5) Both chlorination of the 3 and 4 positions of the aniline moiety and acylanilide bonding were simultaneously required for an acylanilide to produce uncoupling activity. 6) DCMU was less effective than was 3′,4′-DCPA, both in stimulating state 4 and in degenerating the P/O ratio.     

Change of Freezing Resistance and Retention of Metabolic and Differentiation Potentials in Cultured Green Lavandula vera Cells Which Survived Repeated Freeze-thaw Procedures

Cryostorage is one suitable method to preserve various desired types of cells. However, all cells do not survive after storage in liquid nitrogen. This suggests the possibility that the properties of the cells which survive after the storage differ from those of the unfrozen original cells.

Therefore, we did the same freeze-thaw procedure of cultured green Lavandula vera cells over again and compared the metabolic and the differentiation potentials of the cells which survived after the repeated freeze-thaw procedures with those of the unfrozen original cells. The results we found were that the frequency of colony formation of the cells which survived after the procedures was high, but that the biosynthetic capability for biotin and the differentiation potentials such as chloroplast formation and plantlet formation of the cells were equal to those of the unfrozen original cells. Cryostorage of cells in liquid nitrogen is discussed in terms of the preservation of various desired types of cells.     

Quantification of Physical and Cyto-physiological Conditions for the Electrofusion of Saccharomyces cerevisiae

Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 V_p/cm and a DC field of 2 square pulses (7 kV/cm; 60/βsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or l.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 m. The presence of CaCl₂ (up to 0.4 mm) or 0.1 mm CaCl₂ with 0.1 mm MgCl₂ enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28°C.     

Lipid Compositions of Photomixotrophic Green Calluses and Chlorophyll Deficient Leaves of Tobacco

Among photomixotrophic green calluses tested (N. rustica. N. tobacum L. cv. BY-4 and Samsun), the callus of Samsun had the highest contents of chlorophyll and chloroplast lipids, such as monogalactosyldiglyceride (MGDG), digalactosyldiglyceride (DGDG), sulfoquinovosyldigly-ceride (SQDG) and phosphatidylglycerol (PG). However, the chlorophyll and chloroplast lipids in the green callus of Samsun were still 1/6 and 1/3 of that in the parent leaves, respectively. The relative content of a-linolenate in MGDG, DGDG and SQDG of the green calluses were higher than that of the white calluses. The ratios of hexadecatrienoate in MGDG and hexadeceno-ate (Δ³-trans) in PG in the green calluses were trace or less compared with that of the parent leaves. The crude lipids and total fatty acid contents of the chlorophyll deficient leaves (N. taba-cum L. cv. Consolation 402 and Dominant Aurea Su/su) were almost the same as those of the normal leaves (cv. BY-4 and Samsun), although the chlorophyll contents of the chlorophyll deficient leaves were 1/3 ~ 1/4 of that of the normal leaves. The ratios of chloroplast lipids in the total polar lipids in the chlorophyll deficient leaves were a little lower than that in the normal green leaves, but the former had a slightly higher ratio of phospholipids such as phosphatidylcholine and phosphatidylethanolamine than the latter. There were few differences in the fatty acid compositions of each individual lipid betweeen both types of leaves.