Effects of the Complex of Panicum miliaceum Extract and Triticum aestivum Extract on Hair Condition.

Proso millet (Panicum miliaceum L.) and common wheat (Triticum aestivum L.) have been used as major crops in multiple regions since ancient times, and they contain various nutrients that can affect human hair health. This study investigated the various biological effects of a complex of millet extract and wheat extract (MWC) on hair health. Human immortalized dermal papilla cells (iDPCs) for an in vitro study and an anagen-synchronized mouse model for an in vivo study were employed. These findings revealed that the application of the MWC in vitro led to an increase in the mRNA levels of antioxidant enzymes (catalase and SOD1), growth factors (IGF-1, VEGF, and FGF7), and factors related to hair growth (wnt10b, ß-catenin) while decreasing inflammatory cytokine mRNA levels (IL-6 and TNFα). The mRNA levels of hair follicles (HFs) in the dorsal skin of the mouse model in the early and late telogen phases were also measured. The mRNA levels in the in vivo study showed a similar alteration tendency as in the in vitro study in the early and late telogen phases. In this model, MWC treatment elongated the anagen phase of the hair cycle. These findings indicate that the MWC can suppress oxidative stress and inflammation and may elongate the anagen phase by enhancing the growth factors involved in the wnt10b/ß-catenin signaling pathway. This study suggests that the MWC might have significant potential as a functional food for maintaining hair health.

Intertwined Functions of Separase and Caspase in Cell Division and Programmed Cell Death.

Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report a role for the C. elegans separase SEP-1, primarily known for its essential activity in cell division and cortical granule exocytosis, in developmentally programmed cell death when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2 implicating SEP-1 in execution of apoptosis. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation, meiotic chromosome disjunction, egg shell formation, and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors result in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

Methods for evaluating the Caenorhabditis elegans dauer state: standard dauer-formation assay using synthetic daumones and proteomic analysis of O-GlcNAc modifications.

The dauer state is a non-feeding, alternative L3 state characterized by a number of distinctive metabolic and morphological changes. There are many naturally occurring dauer-inducing pheromones, termed daumones, that have been suggested by some to exhibit differences in dauer-inducing activity. Here, we have established a standard dauer-formation assay that uses synthetic daumones 1, 2, and 3, the three major daumones. To analyze the proteome of Caenorhabditis elegans in the dauer state, we focused on O-GlcNAc modification, a cytosolic modification of proteins that is known to interact either competitively or synergistically with protein phosphorylation. Protein O-GlcNAc modification is an important biological process in cells that can ensure the timely response to extracellular stimuli, such as daumone, and maintain cellular homeostasis. Establishing a standard method for assaying dauer formation using different synthetic daumones, and using differences in O-GlcNAcylated proteins during the dauer state to analyze the dauer proteome will lead to a better understanding of dauer biology of C. elegans in the context of animal longevity and adaptation under harsh environments.

STR-33, a novel G protein-coupled receptor that regulates locomotion and egg laying in Caenorhabditis elegans.

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

Identification and characterization of a dual-acting antinematodal agent against the pinewood nematode, Bursaphelenchus xylophilus.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca(2+) channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.

Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis.

Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.

Proteomic analysis of Caenorhabditis elegans.

Proteomic studies of the free-living nematode Caenorhabditis elegans have recently received great attention because this animal model is a useful platform for the in vivo study of various biological problems relevant to human disease. In general, proteomic analysis is carried out in order to address a specific question with respect to differential changes in proteome expression under certain perturbed conditions. In this chapter, we focus on gel-based proteomic analysis of C. elegans subjected to two specific stress conditions during development: induction of the dauer state for whole body protein expression and a temperature shift for egg protein expression. Utilizing these differently perturbed C. elegans protein samples, two-dimensional electrophoresis and differential in-gel electrophoresis methods have led to the discovery of remarkable aspects of the worm's biology. We also provide numerous details about the technical points and protocols necessary for successful experimentation.

Molecular time-course and the metabolic basis of entry into dauer in Caenorhabditis elegans.

When Caenorhabditis elegans senses dauer pheromone (daumone), signaling inadequate growth conditions, it enters the dauer state, which is capable of long-term survival. However, the molecular pathway of dauer entry in C. elegans has remained elusive. To systematically monitor changes in gene expression in dauer paths, we used a DNA microarray containing 22,625 gene probes corresponding to 22,150 unique genes from C. elegans. We employed two different paths: direct exposure to daumone (Path 1) and normal growth media plus liquid culture (Path 2). Our data reveal that entry into dauer is accomplished through the multi-step process, which appears to be compartmentalized in time and according to metabolic flux. That is, a time-course of dauer entry in Path 1 shows that dauer larvae formation begins at post-embryonic stage S4 (48 h) and is complete at S6 (72 h). Our results also suggest the presence of a unique adaptive metabolic control mechanism that requires both stage-specific expression of specific genes and tight regulation of different modes of fuel metabolite utilization to sustain the energy balance in the context of prolonged survival under adverse growth conditions. It is apparent that worms entering dauer stage may rely heavily on carbohydrate-based energy reserves, whereas dauer larvae utilize fat or glyoxylate cycle-based energy sources. We created a comprehensive web-based dauer metabolic database for C. elegans (www.DauerDB.org) that makes it possible to search any gene and compare its relative expression at a specific stage, or evaluate overall patterns of gene expression in both paths. This database can be accessed by the research community and could be widely applicable to other related nematodes as a molecular atlas.

Effects of sterols on the development and aging of Caenorhabditis elegans.

Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.

Proteomic analysis of the sterol-mediated signaling pathway in Caenorhabditis elegans.

Since Caenorhabditis elegans is incapable of de novo cholesterol biosynthesis, it must utilize other nonpermissive sterols that are present in the environment by converting them into cholesterol for cellular function. The inhibition of sterol conversion to cholesterol in C. elegans by various sterol biosynthesis inhibitors (SBIs) is known to cause serious defects in the development of these worms. To determine the biochemical consequences of these physiological abnormalities, one can perform a proteomic analysis of worms of a certain stage that are grown in the presence of SBIs in order for the differential expression of proteins involved in the sterol-mediated signaling pathway to be identified. For example, reductions in the expression of lipoprotein family members, such as vitellogenin-2 and vitellogenin-6, are prominent in azacoprostane-treated worms. This phenomenon is also seen in worms treated with AY-9944, which blocks the conversion of 7-dehydrocholesterol, a major sterol present in C. elegans, to cholesterol.

The efficiency of RNA interference in Bursaphelenchus xylophilus.

RNA interference (RNAi) was performed on several essential genes in the pinewood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. Double-stranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reverse-transcription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAi phenotypes for essential genes was relatively low but consistent, indicating that RNAi should be useful for studying the in vivo functions of B. xylophilus gene products.

C. elegans: an invaluable model organism for the proteomics studies of the cholesterol-mediated signaling pathway.

With the availability of its complete genome sequence and unique biological features relevant to human disease, Caenorhabditis elegans has become an invaluable model organism for the studies of proteomics, leading to the elucidation of nematode gene function. A journey from the genome to proteome of C. elegans may begin with preparation of expressed proteins, which enables a large-scale analysis of all possible proteins expressed under specific physiological conditions. Although various techniques have been used for proteomic analysis of C. elegans, systematic high-throughput analysis is still to come in order to accommodate studies of post-translational modification and quantitative analysis. Given that no integrated C. elegans protein expression database is available, it is about time that a global C. elegans proteome project is launched through which datasets of transcriptomes, protein-protein interaction and functional annotation can be integrated. As an initial target of a pilot project of the C. elegans proteome project, the cholesterol-mediated signaling pathway will be an excellent example since, like in other organisms, it is one of the key controlling pathways in cell growth and development in C. elegans. As this field tends to broaden to functional proteomics, there is a high demand to develop the versatile proteome informatics tools that can mange many different data in an integrative manner.

Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone.

Pheromones are cell type-specific signals used for communication between individuals of the same species. When faced with overcrowding or starvation, Caenorhabditis elegans secrete the pheromone daumone, which facilitates communication between individuals for adaptation to adverse environmental stimuli. Daumone signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended life. Because daumone is a key regulator of chemosensory processes in development and ageing, the chemical identification of daumone is important for elucidating features of the daumone-mediated signalling pathway. Here we report the isolation of natural daumone from C. elegans by large-scale purification, as well as the total chemical synthesis of daumone. We present the stereospecific chemical structure of purified daumone, a fatty acid derivative. We demonstrate that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. These results should help to elucidate the daumone-mediated signalling pathway, which might in turn influence ageing and obesity research and the development of antinematodal drugs.

Requirement of tyrosylprotein sulfotransferase-A for proper cuticle formation in the nematode C. elegans.

Tyrosine O-sulfation is one of the post-translational modification processes that occur to membrane proteins and secreted proteins in eukaryotes. Tyrosylprotein sulfotransferase (TPST) is responsible for this modification, and in this report, we describe the expression pattern and the biological role of TPST-A in the nematode Caenorhabditis elegans. We found that TPST-A was mainly expressed in the hypodermis, especially in the seam cells. Reduction of TPST-A activity by RNAi caused severe defects in cuticle formation, indicating that TPST-A is involved in the cuticle formation in the nematode. We found that RNAi of TPST-A suppressed the roller phenotype caused by mutations in the rol-6 collagen gene, suggesting that sulfation of collagen proteins may be important for proper organization of the extracellular cuticle matrix. The TPST-A RNAi significantly decreased the dityrosine level in the worms, raising the possibility that the sulfation process may be a pre-requisite for the collagen tyrosine cross-linking.