In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31 ؊ ;CD146 ؊ SP cells, demonstrating CD34 ؉ and vascular endothelial growth factor-2 (VEGFR2)/Flk1 ؉ , were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

The activities to induce TNF-α production by a monocytic cell line, THP-1, and ICAM-1 expression and IL-6 production by human gingival fibroblasts were detected in plural membrane lipoproteins of Mycoplasma salivarium. Although SDS-PAGE of the lipoproteins digested by proteinase K did not reveal any protein bands with molecular masses higher than approximately10 kDa, these activities were detected in the front of the gel. A lipoprotein with a molecular mass of 44 kDa (Lp44) was purified. Proteinase K did not affect the ICAM-1 expression-inducing activity of Lp44, but lipoprotein lipase abrogated the activity. These results suggested that the proteinase K-resistant and low molecular mass entity, possibly the N-terminal lipid moiety, played a key role in the expression of the activity. The N-terminal lipid moiety of Lp44 was purified from Lp44 digested with proteinase K by HPLC. Judging from the structure of microbial lipopeptides as well as the amino acid sequence and infrared spectrum of Lp44, the structure of the N-terminal lipid moiety of Lp44 was speculated to be S-(2, 3-bisacyloxypropyl)-cysteine-GDPKHPKSFTEWV-. Its analogue, S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF, was synthesized. The lipopeptide was similar to the N-terminal lipid moiety of Lp44 in the infrared spectrum and the ICAM-1 expression-inducing activity. Thus, this study suggested that the active entity of Lp44 was its N-terminal lipopeptide moiety, the structure of which was very similar to S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam 2 CG DPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentansderived lipopeptide MALP-2 (Pam 2 CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-B reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-B-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.Various bacterial cell wall components such as lipopolysaccharides (LPS), lipoteichoic acid, peptidoglycans, and lipoproteins (LP) have been shown to activate macrophages, fibroblasts, or lymphocytes to induce production of cytokines (16). Escherichia coli LP were first characterized and sequenced by Braun (9), and they have been demonstrated to be biologically active (5)(6)(7)(8)20). The part of LP responsible for biological activity is the N-terminal lipopeptide moiety, the structure of which is S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-SerSer-Asp-Ala-(Pam 3 CSNNA-) (7).Mycoplasmas, wall-less microorganisms, also possess LP capable of activating macrophages or fibroblasts (11,27,28,31,32). Mühlradt et al. (27,28) recently identified a 2-kDa lipopeptide, MALP-2, from Mycoplasma fermentans that is capable of activating monocytes/macrophages, and these authors determined the structure to be S-(2,3-bispalmitoyloxypropyl) Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys (Pam 2 CGNNDESNISFKEK). We have also found that Mycoplasma salivarium LP activate normal human gingival fibroblasts (HGF) to induce production of inflammatory cytokines and surface expression of ICAM-1 and have purified a 44-kDa LP (LP44) responsible for the activity (32). The structure of the N-ter...

This investigation demonstrates the potential utility of fractionated SP cells as a source of cells for total pulp regeneration complete with angiogenesis and vasculogenesis.

Our data show that NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-beta signalling through a cGMP-dependent pathway. Our findings suggest that NO may play a beneficial role in atherogenesis in part by limiting vascular calcification.

SummaryMycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Tolllike receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF-k k k k B, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid. The activity was synergically augmented by co-expression of TLR6, but not by co-expression of other TLRs. Under the condition of co-expression of TLR2 and TLR6, the lipoproteins could induce maximum NF-k k k k B activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant-negative forms of MyD88 and FADD, but not IRAK-4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant-negative forms also downregulated the activation of both NF-k k k k B and caspase-8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2-and TLR6-mediated sequential bifurcate responses: NF-k k k k B activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.