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.

Oxygen saturation is associated with recurrent primary spontaneous pneumothorax treated by intercostal chest drainage.

BACKGROUND: Primary spontaneous pneumothorax (PSP) is a condition that may lead to acute chest pain or dyspnea on exertion. Treatment with an intercostal chest drainage (ICD) is warranted. There is limited data on risk factors of recurrent PSP in patients treated with the ICD alone. This study aimed to evaluate risk factors of recurrent PSP in patients with PSP and treated with the ICD. METHODS: This was a retrospective study and enrolled patients diagnosed as PSP and treated with an ICD. Eligible patients were divided into two groups by evidence of recurrent PSP. Baseline characteristics, physical signs, laboratory results, and duration of ICD treatment were studied and recorded from medical charts. Factors associated with recurrent PSP were computed by using multivariate logistic regression analysis. RESULTS: There were 80 patients met the study criteria. Of those, 21 patients (26.3%) had recurrent PSP. Of those, 21 patients (26.3%) had recurrent PSP. There were eight factors in the final model for recurrent PSP. Only oxygen saturation at the time of diagnosis was independently associated with recurrent PSP. The adjusted odds ratio (95% confident interval) was 0.57 (0.34, 0.96). A cut point of 96% of oxygen saturation gave sensitivity of recurrent PSP of 80.95%. CONCLUSION: The prevalence of recurrent PSP was 26.3% in patients with PSP and treated with the ICD. Initial oxygen saturation may be an indicator for recurrent PSP.

Factors Associated with Duration of Intercostal Chest Drainage in Patients with Primary Spontaneous Pneumothorax and the Role of Pulmonary Rehabilitation.

PURPOSE: Primary spontaneous pneumothorax (PSP) is an urgent/emergency condition. Treatment with intercostal chest drainage (ICD) is necessary, particularly in symptomatic patients or those with tension. A previous study found that systematic breathing exercise significantly reduced ICD duration when compared with controls. This study aimed to evaluate if pulmonary rehabilitation can reduce the duration of ICD treatment in patients with PSP. PATIENTS AND METHODS: This was a retrospective study of patients diagnosed with PSP treated with ICD. Duration of ICD treatment was recorded from patients' medical charts. Factors associated with ICD duration were calculated using linear regression analysis. RESULTS: There were 66 patients who met the study criteria, with average (SD) age and body mass index of 31.68 (13.53) years and 20.94 (2.72) kg/m2. The majority of the patients were male (72.73%), and average (SD) duration of ICD treatment was 9.90 (7.83) days. Three factors remained in the final model: body mass index, systolic blood pressure, and recurrent PSP. Two factors were independently associated with longer ICD duration: systolic blood pressure and recurrent PSP, with adjusted coefficients of 0.21 (p value 0.041) and 7.69 (p value 0.039), respectively. Pulmonary rehabilitation was not included in the final model. CONCLUSION: Patients with a history of recurrent PSP or high systolic blood pressure at presentation may require longer ICD duration. Pulmonary rehabilitation was not associated with the duration of ICD treatment.

Expression of endogenous phospholipase D1, localized in mouse submandibular gland, is greater in females and is suppressed by testosterone.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4 weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2 weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

Localization of phospholipase C ß3 in the major salivary glands of adult mice.

Phospholipase C (PLC)ß has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCß isoforms in mouse salivary glands in situ. A single major band was detected for PLCß3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCß1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCß3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCß3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.

Expression and localization of VIAAT in distal uriniferous tubular epithelium of mouse.

Vesicular inhibitory amino acid transporter (VIAAT) is a transmembrane transporter which is responsible for the storage of gamma-aminobutyric acid (GABA) or glycine in synaptic vesicles. According to recent studies, GABA is known to be expressed in the kidney. For clear understanding of the intra-renal GABA signaling, the localization of VIAAT was examined in the present study. Intense immunoreactivity was found largely confined to the distal tubule epithelia, especially distinct in the inner medulla, although the immunoreactivity was discerned more or less in all tubules and glomeruli. No distinct immunoreactivity was seen in capillary endothelia or interstitial fibroblasts. In immuno-DAB and immuno-gold electron microscopy, the immunoreaction was found at the basal infoldings of plasma membranes and basal portions of the lateral plasma membranes, but not in any vesicles or vacuoles within the distal tubular cells. The significance of the enigmatic finding, localization of a vesicular molecule on selected portions of the plasma membrane of distal tubular cells, was discussed in view of the possibility of paracrine or autocrine effects of GABA on some other uriniferous tubular cells or interstitial cells.

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol.

OBJECTIVE: We wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists. MATERIALS AND METHODS: SMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. RESULTS: Selective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol. CONCLUSION: The present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors.

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.

Identification in immuno-electron microscopy of solitary multi-polar peripheral neurons of adult Opisthorchis viverrini by antibody against rat diacylglycerol kinase / Identificación por microscopía imuno-electrónica de las neuronas periféricas multipolares solitarias del Opisthorchis viverrini adulto mediante anticuerpo contra la diacilglicerol kinasa de ratas

By utilizing the antibody for rat DGKz a substantial number of immunopositive cells were found in the OV (Opisthorchis viverrini). The immunopositive cells appeared solitarily and they were distributed rather symmetrically to the longitudinal axis of the OV. Some of them were located in close proximity to internal organs such as uterus, ovary, testes, vitelline glands and guts. The immunostained cells extended tapering processes horizontally or obliquely to the OV longitudinal axis. In immuno-electron microscopy, the immunopositive cells were characterized by intensely immunostained mitochondria and weakly immunostained cytoplasm and immunonegative chromatin-poor nucleus. Vacuoles of various sizes without the immunoreactivity were also contained in the cells. Thin cellular processes without the immunoreactivity were found to enclose thinly the entire surfaces of the immunostained cells and processes, and they were in continuity with the interstitial partition-like processes which contained nuclei and aggregation of microfibrils at some distance from the cytoplasmic envelopes. The present finding suggests the possibility that the immunostained cells were peripheral neurons enveloped by peripheral glia and that the glia are of mesenchymal origin because of their cytoplasmic continuity to the interstitial partition-like processes. The motor or sensory nature of the neurons remains to be elucidated.

Mediante el uso del anticuerpos DGK para rata se determinó un número considerable de células inmunopositivas en el Opisthorchis viverrini (OV). Las células inmunopositivas aparecían solitarias y se distribuían simétricamente al eje longitudinal de la OV. Algunas estaban ubicadas en las proximidades de los órganos internos como el útero, ovarios, testículos, glándulas vitelinas e intestino. Las células inmunoteñidas extendían sus procesos horizontalmente u oblicuamente al eje longitudinal de la OV. Por microscopía inmunoelectrónica, las células inmunopositivas se caracterizaron por presentar mitocondrias intensamente teñidas, citoplasma con tinción débil e inmunonegatividad en núcleos pobres en cromatina. También se observó en las células, vacuolas de diversos tamaños sin inmunorreactividad. Se encontraron procesos celulares sin inmunorreactividad para cerrar finamente todas las superficies de las células y procesos, y se continuaron con los procesos de partición intersticiales que contenían núcleos y agregación de microfibrillas a cierta distancia de las envolturas citoplásmicas. El presente hallazgo sugiere la posibilidad de que las células inmunoteñidas son neuronas periféricas envueltas por glia periférica y que la glía presenta origen mesenquimal debido a su continuidad citoplasmática con los procesos de partición intersticiales. La naturaleza motora o sensorial de las neuronas aún no se ha dilucidado.

Selective localization of diacylglycerol kinase (DGK)ζ in the terminal tubule cells in the submandibular glands of early postnatal mice.

The present immunohistochemical study was attempted to localize in the submandibular glands of mice at various postnatal stages a diacylglycerol kinase (DGK) isoform termed DGKζ which is characterized by a nuclear localization signal and a nuclear export signal. This attempt was based on following facts: the continuous postnatal differentiation of glandular cells in the rodent submandibular gland, the regulatory role of DGK in the activity of protein kinase C (PKC) through attenuation of diacylglycerol (DAG), and the possible involvement of PKC in various cellular activities including the saliva secretion as well as the cell differentiation. As a result, a selective localization of immunoreactivity for DGKζ was detected in terminal tubule (TT) cells which comprise a majority of the newborn acinar structure and differentiate into the intercalated duct cells and/or the acinar cells. The immunoreactivity was deposited in portions of the cytoplasm lateral and basal to the nucleus, but not in the nuclei themselves. Although the immunoreactive TT cells remained until later stages in female specimen than in male, they eventually disappeared in both sexes by young adult stages. The present finding suggests that the regulatory involvement of DGKζ in PKC functions via control of DAG is exerted in the differentiation of the TT cells. In addition, another possible involvement of DGKζ in the regulation of secretion of the TT cells as well as its functional significance of its nuclear localization in the submandibular ganglion cells was also discussed.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

BACKGROUND: Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. RESULTS: A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. CONCLUSIONS: Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.