Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

Low molecular weight sulfated galactan (LMSG) supplemented with octanoyl ester (Oct-LMSG) demonstrated superior wound healing activity compared to the unsupplemented LMSG in a fibroblast wound model. To test the hypothesis that the increased bioactivity of Oct-LMSG may depend on its penetration into the plasma membrane, its cellular uptake was investigated and collagen production in fibroblast cells was assessed for the first time. The cellular uptake of Oct-LMSG was examined using indirect immunofluorescence and a confocal laser scanning microscope. In addition, the degree of fibroblast activation associated with this uptake was evaluated. The results indicated increased LMSG internalization in fibroblasts treated with Oct-LMSG. Transmission electron micrographs revealed the ultrastructure of active protein production in fibroblasts upon treatment with Oct-LMSG. In addition, Oct-LMSG upregulated the expression of type I collagen mRNA and proteins, as well as related signaling molecules involved in collagen synthesis, including collagen type I α1 chain (Col1A1), Col1A2, phosphorylated (p)-Smad2/3 and p-Smad4. The current findings support the notion that the supplementation of LMSG with octanoyl enhanced its cellular uptake into fibroblasts and, as a result, regulated the expression of type I collagen in fibroblasts via the activation of the Smad signaling pathway. This study demonstrates the therapeutic potential of Oct-LMSG in promoting tissue regeneration.

Sulfated Galactans from Gracilaria fisheri with Supplementation of Octanoyl Promote Wound Healing Activity In Vitro and In Vivo.

Sulfated galactans (SG) isolated from Gracilaria fisheri is partially degraded (DSG), and subsequentially supplemented with octanoyl (DSGO) and sulfate (DSGS) groups. The molecular weights of DSG, DSGO, and DSGS are 7.87, 152.79, and 97.07 kDa, respectively. The modification is confirmed using FTIR and NMR, while in vitro wound healing activity is assessed using scratched wound fibroblasts. The results reveal that DSGO exhibits highest percentage of wound closure in scratched fibroblast L929 cells. Furthermore, DSGO is able to promote proliferation and accelerate migration of scratched fibroblasts, which correspond to the regulation of proteins and mRNA (Ki67, p-FAK, vimentin, and E-cadherin) determined by Western blotting and qPCR analysis. The superior wound healing activity of DSGO is also confirmed in excision wound of rats. The results demonstrate that DSGO significantly enhances the percentage of wound closure, re-epithelialization, and collagen arrangement, increases α-smoth muscle actin (α-SMA) and vimentin expression, and decreases that of tumor necrosis factor-α (TNF-α) at the wound site. The results suggest that degraded SG supplemented with medium-chain fatty acids of octanoyl group may pass through the membrane, subsequently activating the mediators associated with proliferation and migration of fibroblasts, which can potentially lead to the promotion of wound healing activity.

Increased Sulfation in Gracilaria fisheri Sulfated Galactans Enhances Antioxidant and Antiurolithiatic Activities and Protects HK-2 Cell Death Induced by Sodium Oxalate.

Urolithiasis is a common urological disease characterized by the presence of a stone anywhere along the urinary tract. The major component of such stones is calcium oxalate, and reactive oxygen species act as an essential mediator of calcium oxalate crystallization. Previous studies have demonstrated the antioxidant and antiurolithiatic activities of sulfated polysaccharides. In this study, native sulfated galactans (N-SGs) with a molecular weight of 217.4 kDa from Gracilaria fisheri were modified to obtain lower molecular weight SG (L-SG) and also subjected to sulfation SG (S-SG). The in vitro antioxidant and antiurolithiatic activities of the modified substances and their ability to protect against sodium oxalate-induced renal tubular (HK-2) cell death were investigated. The results revealed that S-SG showed more pronounced antioxidant activities (DPPH and O2- scavenging activities) than those of other compounds. S-SG exhibited the highest antiurolithiatic activity in terms of nucleation and aggregation, as well as crystal morphology and size. Moreover, S-SG showed improved cell survival and increased anti-apoptotic BCL-2 protein in HK-2 cells treated with sodium oxalate. Our findings highlight the potential application of S-SG in the functional food and pharmaceutical industries.

Structural characterization, antioxidant activity, and protective effect against hydrogen peroxide-induced oxidative stress of chemically degraded Gracilaria fisheri sulfated galactans.

Sulfated polysaccharides (SPs) possess an extensive range of biological activities, such as the inhibition of oxidation, correlated with their molecular weight (MW) and chemical structure. In this study, we used the trifluoroacetic acid (TFA) controlled degradation method to degrade sulfated galactans (SG) isolated from Gracilaria fisheri and evaluated the antioxidant and protective effects of the low molecular weight SG (LMSG) against H2O2 on fibroblast cells for the first time. Degradation of native SG (NSG) with an initial MW of 217.45 kDa using different concentrations of TFA resulted in five degraded NSG with MW of 97.23, 62.26, 30.74, 2.63, and 2.59 kDa. The reduction in MW was positively correlated with TFA concentrations. Chemical structure analyses using FTIR and NMR indicated that the TFA degradation process did not significantly change the LMSG polysaccharide main chain but did change the functional groups. LMSG exhibited higher scavenging activities and enhanced the cellular activities of GSH, CAT, and SOD enzymes. Moreover, LMSG activated Nrf-2/ARE signaling and increased expression of antioxidant genes CAT and SOD, which corresponded to increased protective effects against H2O2-induced ROS generation in fibroblast cells. The study reveals modification of NSG by acid TFA degradation resulted in the creation of LMSG, which showed greater antioxidant activity.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.

Sexual dimorphism using the interstyloid distances and clinical implication for elongated styloid process in Northeastern Thailand / Dimorfismo sexual usando la distancia inter-estiloide y la implicación clínica del proceso estiloides elongado en el Noreste de Tailandia

Skeletal remains are crucial in forensic identification of the sex, especially human skulls including the styloid process, a bony projection from the skull. Hence, the objectives of the present study were undertaken to assess the value of the styloid process for the sex identification of unknown skulls and also to investigate the prevalence of elongated styloid process in 102 human dry skulls from the northeast Thai population. As a result, the interstyloid distances at both base and tip of the styloid processes were found to be significantly different between male and female specimens, although no significant difference was found in the length of the styloid process between males and females. In addition, the occurrence of the elongated styloid process was not associated with the gender, although its prevalent laterality on the left was recognized. It is suggested that the styloid process can be applied to the sex identification by measuring the interstyloid distance at the base or the tip of these processes.

Los restos óseos son cruciales para la identificación forense del sexo, especialmente en los cráneos humanos, incluyendo el proceso estiloides, una proyección ósea del cráneo. Por lo tanto, los objetivos del presente estudio consistieron en evaluar el valor del proceso estiloides en la identificación del sexo de cráneos desconocidos y también para investigar la prevalencia del proceso estiloides elongado en 102 cráneos secos humanos de la población del Noreste de Tailandia. Como resultado, se encontró que las distancias inter-estiloides tanto en la base y la punta de los procesos estiloides eran significativamente diferentes entre las muestras de hombres y mujeres, aunque no se encontró diferencia significativa en la presencia del proceso estiloides entre ambos. Además, la aparición del proceso estiloides elongado no se asoció con el sexo, aún cuando se observó su prevalencia en el lado izquierdo. Sugerimos que el proceso estiloides se puede utilizar en la identificación del sexo mediante la medición de la distancia inter-estiloide en la base o en la punta de estos procesos.

Anti-Tumor and Immune Enhancing Activities of Rice Bran Gramisterol on Acute Myelogenous Leukemia.

BACKGROUND: Acute myelogenous leukemia (AML) is a cancer of the blood that most commonly affects human adults. The specific cause of AML is unclear, but it induces abnormality of white blood cells that grow rapidly and accumulate in bone marrow interfering with the production and functions of the normal blood cells. AML patients face poor prognosis and low quality of life during chemotherapy or transplantation of hematopoietic stem cells due to the progressive impairment of their immune system. The goal of this study is to find natural products that have the potential to delay growth or eliminate the abnormal leukemic cells but cause less harmful effect to the body's immune system. METHODS AND FINDINGS: The unsaponified fraction of Riceberry rice bran (RBDS) and the main pure compound, gramisterol, were studied for cytotoxicity and biological activities in WEHI-3 cells and in the leukemic mouse model induced by transplantation of WEHI-3 cells intraperitoneally. In the in vitro assay, RBDS and gramisterol exerted sub-G1 phase cell cycle arrest with a potent induction of apoptosis. Both of them effectively decreased cell cycle controlling proteins (cyclin D1 and cyclin E), suppressed cellular DNA synthesis and mitotic division, and reduced anti-apoptosis Bcl-2 protein, but increased apoptotic proteins (p53 and Bax) and activated caspase-3 enzyme in the intrinsic cell death stimulation pathway. In leukemic mice, daily feeding of RBDS significantly increased the amount of immune function-related cells including CD3+, CD19+, and CD11b+, and elevated the serum levels of IFN-γ, TNF-α, IL-2, and IL-12ß cytokines, but suppressed IL-10 level. At the tumor sites, CD11b+ cells were polarized and became active phagocytotic cells. Treatment of mice normal immune cells with gramisterol alone or a combination of gramisterol with cytokines released from RBDS-treated leukemic mice splenocytes culture synergistically increased pSTAT1 transcriptional factor that up-regulated the genes controlling cell survival and function. Phosphorylation of STAT1 was absent in WEHI-3. Instead, similar treatments significantly decreased pSTAT3 signaling that regulates transcription of genes controlling tumor growth and proliferation. CONCLUSIONS: Rice bran gramisterol possesses a promising anti-cancer effect against a tumor of white blood cells and induces the production of anti-cancer immune-related cytokines. Gramisterol induces cell cycle arrest and apoptosis via suppression of pSTAT3 signaling control of tumor cells' growth and progression. Gramisterol increased IFN-γ production and prevented the dysfunctional immune system of leukemic mice by enhancing pSTAT1 transcription signal controlling proliferation and functions of hematopoietic cells in the spleen. Together with IFN-γ, gramisterol efficiently facilitates leukemic mice immune system modulation leading to improvement of the AML condition. Administration of RBDS containing gramisterol potentiates immune recovery of leukemic mice and extends their survival. This finding encourages the medicinal application of rice bran gramisterol as a palliative treatment or an alternative agent for future drug development against AML.