Praphaporn Stewart

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1-3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.

Ultrastructure of differentiating oocytes and vitellogenesis in the giant freshwater prawn, Macrobrachium rosenbergii (De Man).

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone‐shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc1) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc2) show nuclear heterochromatin with a “clock face” pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc2. Early vitellogenic oocytes (Oc3) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc4) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc3 and Oc4, but there may be some undetected exogenous Vg production. Microsc. Res. Tech. 2012.

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ-sinus gland (XO-SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO-SG, raises new questions concerning the established mode of MIH action.

Antioxidant activity and ultrastructural changes in gastric cancer cell lines induced by Northeastern Thai edible folk plant extracts

BackgroundPhytochemical products have a critical role in the drug discovery process. This promising possibility, however, necessitates the need to confirm their scientific verification before use. Hence, this study aims to evaluate (1) the antioxidant activity, (2) cytotoxicity potential, and (3) the effect on ultrastructural alteration in gastric cancer cell lines through exposure to fractions of three local Northeastern Thai edible plants.MethodsPlants, Syzygium gratum, Justicia gangetica and Limnocharis flava were extracted with ethyl acetate, and each crude extract analysed for their total phenolics content by Folin-Ciocalteu method. Their antioxidant activity was assessed using the ABTS system. The extracts were then assayed for cytotoxicity on two gastric cancer cell lines Kato-III and NUGC-4, and compared with Hs27 fibroblasts as a control using the MTT assay. The cell viability (%), IC50 values, as well as the ultrastructural alterations were evaluated after treatment with one way analysis of variance (ANOVA).ResultsThe total phenolic values of the ethyl acetate extracts were well correlated with the antioxidant capacity, with extracted product of S. gratum displaying the highest level of antioxidant activity (a 10-fold greater response) over J. gangetica and L. flava respectively. Exposure of S. gratum and J. gangetica extracts to normal cell lines (Hs27) resulted in marginal cytotoxicity effects. However, through a dose-dependent assay S. gratum and J. gangetica extracts produced cytotoxicological effects in just over 75 percent of Kato-III and NUGC-4 cell lines. In addition, apoptotic characteristic was shown under TEM in both cancer cell lines with these two extracts, whereas characteristics of autophagy was found in cell lines after post exposure to extracts from L. flava.ConclusionsFrom these three plants, S. gratum had the highest contents of phenolic compounds and antioxidant capacity. All of them found to contain compound(s) with cytotoxicity in vitro on cancer cells but not on normal cell lines as resolved in tissue culture and ultrastructural analysis. This is the first report to show the effect on cellular alteration as apoptosis of an ethyl acetate extract of S. gratum and J. gangetica. Further studies are now focused on individual isolates and their function, prioritizing on S. gratum and J. gangetica for the development of novel therapeutics and combatants against cancer.

Histological studies of the ovaries of two tropical portunid crabs, Portunus pelagicus (L.) and Scylla serrata (F.)

Summary The histology of ovaries of adult crabs, Portunus pelagicus and Scylla serrata, were studied and compared using light microscopy. In general, the histological features, the steps of oogenetic cells and the stages of ovarian development appear similar in the two species. The ovary possesses three to four lobes, and each lobe is divided into lobules. A thick capsule surrounds the ovarian lobes and its extensions cover and separate each lobule. The lobule is composed of follicles, each containing a cluster of oocytes at different steps within the confinement of connective tissue trabeculae. There are five steps of oogenetic cells, i.e., oogonia (Og) and four steps of oocytes (Oc1, Oc2, Oc3, Oc4). Og are the earliest germ cells and are located in the center of each lobule. They divide mitotically and move into each follicle where they become Oc1. Oc2 rapidly increase in size, exhibits a clockface nucleus and basophilic cytoplasm. Oc2 possess larger nuclei, non-condensed chromatin, a prominent nucleolus and are intensely basophilic. Oc1 and Oc2 are previtellogenic and do not contain yolk granules. Oc3 are larger in size, with a slate grey cytoplasm and contain a few yolk granules. Oc4 are fully mature oocytes, in possession of completely dispersed chromatin in the nucleus and a very prominent nucleolus. The cytoplasm is eosinophilic due to the presence of numerous yolk granules. The Periodic acid-Schiff reaction and Sudan black stains suggest that Oc4 also contain large amounts of glycoprotein and lipid in their cytoplasm. The ovarian cycle is divided into four stages, i.e., spawn-spent (stage I), proliferative (stage II), pre-mature (stage III) and mature (stage IV). Each stage contains different cellular associations: stage I is characterized by the reconfiguration of connective tissue scaffold, plus remnants of unspawned oocytes; stage II contains numerous dividing Og and a few Oc1; stage III contains primarily Oc1, Oc2, Oc3; and stage IV contains mostly Oc4 and small clusters of Og.

Distribution of Gaba in the Nerve Ganglia of Haliotis asinina Linnaeus

ABSTRACT Gamma-aminobutyric acid (GABA) is a major neurotransmitter and effective settlement inducer in abalone aquaculture. This study aimed to explore the distribution of GABA within neural tissues of Haliotis asinina. Gamma-aminobutyric acid was found in neuronal cell type 1 of 3 major ganglia (i.e., cerebral, pleuropedal, and visceral ganglia) of both sexes. The distribution of GABA-immunoreactive (-ir) cells in the cerebral ganglion was concentrated mostly in the cortex region of the dorsal horn, whereas it was scattered throughout the pleuropedal ganglion, with more in the upper half. Gamma-aminobutyric acid-ir nerve fibers were found throughout the neuropils of the ganglia. The visceral ganglion had the least numbers of GABA-ir neurons compared with the other 2 ganglia. The cells were distributed mainly in the dorsal horn. We also observed GABA to be colocalized with 2 other neurotransmitters: serotonin (5-HT) and dopamine (DA). In the cerebral ganglion, fluorescence double staining of GABA and 5-HT, and GABA and DA showed immunoreactivity in separate cells and was also colocalized in the same cells. In the pleuropedal ganglion, the staining pattern was similar to the cerebral ganglion, but positive-staining cells were less numerous. In the visceral ganglion, GABA and DA, and GABA and 5-HT were colocalized in the same cell types. Overall, we found that GABAergic cells were most numerous in the cerebral ganglion of H. asinina. Further studies are required to determine the functions of these neurotransmitters in relation to their distribution.

The Tetrapeptide Apgw-Amide Induces Somatic Growth in Haliotis asinina Linnaeus

ABSTRACT APGW-amide is a well-known neurohormone modulator in several molluscs, and is involved in motor activities, feeding, and sexual behavior. In this report we show that injections of APGW-amide into 4-mo-old juvenile Haliotis asinina stimulate growth of body weight and, to a lesser degree, shell length. The injections were given at 0 (control), 20, and 200 ng/g body weight (BW), at 1-wk intervals for 14 wk. BW and shell length (SL) were measured every week, and growth rates were calculated. When compared with control animals, there was an approximate 2-fold increase in body growth rates of animals given 20 ng/g BW and 200 ng/g BW APGW-amide (P ≤ 0.05), whereas only 20 ng/g BW APGW-amide produced significantly greater SL than controls (P ≤ 0.05), with an approximate 1.2-fold increase. Using an immunoperoxidase technique, we showed the presence of APGW-amide in neuronal cells of the cerebral ganglia and nerve fibers. Overall, these data indicate that APGW-amide is an important neurohormone/neuromodulator in the nervous system of H. asinina and plays a role in controlling the body growth of H. asinina.

Antimicrobial activities of Cantareus (Helix) aspersa mucus

T aim of this study was to evaluate the bioequivalence of a fixed-dose repaglinide/metformin combination (FDC) tablet at a dose of 2/500 mg with co-administration of equivalent doses of repaglinide (2 mg) and metformin (500 mg) as individual (EDI) tablets in healthy Korean male volunteers.This study was conducted as an open-label, randomized, single-dose, 2-period 2-squence crossover design in 48 healthy Korean male volunteers who received an FDC tablet or EDI tablets after a 12-hour overnight fast in each period. Plasma concentrations of repaglinide and metformin up to 24 hours were determined using a UPLC-MS/MS method. The pharmacokinetic parameters such as AUC0-t, AUC0-∞, Cmax, Tmax and t1/2, were analyzed. Analysis of variance was carried out using logarithmically transformed AUC0-t, AUC0-∞ and Cmax. The formulations were considered bioequivalent if the log-transformed ratios of AUC0–t, AUC0–∞, and Cmax were within the predetermined bioequivalence range (80-125%) established by the US Food and Drug Administration. Tolerability was assessed throughout the study. No significant sequence effect was detected. The point estimates (90% CIs) for AUC0-t, AUC0-∞ and Cmax based on EDI tablets were 1.101 (1.023-1.185), 1.099 (1.022-1.184) and 1.126 (1.015-1.249) for repaglinide, and 0.952 (0.896-1.011), 0.950 (0.897-1.007) and 0.984 (0.927-1.045) for metformin, respectively, satisfying the bioequivalence criteria of 80-125% as proposed by the US FDA. This single-dose study found that both Repaglinide and Metformin in a fixed-dose combination tablet were bioequivalent to individual tablets of repaglinide 2 mg and metformin 500 mg in these fasting, healthy Korean male volunteers.I resistance plays important roles during the initiation and pathogenesis of the disease. Thus, treatment of T2DM targeted on insulin resistance is one of the major strategies. Unfortunately, current clinical insulin sensitizer agent thiazolidinediones (TZDs), which are validated to be potent agonist of nuclear receptor PPARγ, are beset by adverse side effects evoked by full PPARγ agonism. Aimed to develop the safe and efficient insulin sensitizer, researchers proposed the concept of selective PPARγ modulator (sPPARγM), which is believed to retain potent insulin sensitizing activity yet minimize side effects derived from full PPARγ agonism. However, the sPPARγM developed slowly because of the tardiness of the mechanism on the selective modulation of PPARγ. Recent studies demonstrated that liands activated PPARγ mediated insulin sensitizing effect dependent on the inhibition of CDK5 mediated phosphorylation at serine 273 of PPARγ (pSer273PPARγ) in adipose depots but independent on classical full agonism related transcriptional activity, which provides an explicit avenue to develop novel sPPARγM. In this study, we found that a novel non-TZD compound L312 interacts with PPARγ. Evaluation of activity indicated that L312 showed equal binding affinity with pioglitazone to PPARγ but displayed a very different modulation of PPARγ activity. In db/db mice, L312 considerably improve insulin resistance and lipid variables compared to TZD, yet with reduced side effects such as weight gain and fluid retention. Molecular mechanism revealed that L312 effectively inhibited pSer273PPARγ and exerted a selective gene expression profile in epididymal WAT. In conclusion, we determined that L312 is novel sPPARγM with potent inhibition of pSer273PPARγ and suggested that L312 may represent a novel template for designing sPPARγM with advantages over current TZDs.R developments in cancer biology have identified the existence of a sub-population of cells—cancer stem cells—that are immune to most traditional therapies (e.g., chemotherapy and radiotherapy) and have the ability to repair their damaged DNA. Here, we show the resistance of hepatocarcinoma stem cells and glioblastoma multiform stem cells to both radiation and therapy. Also, we show the efficiency of the conjugated iron oxide nanoparticles for the in vivo disruption of Notch signaling by the gamma secretase inhibitor DAPT [N-(N-((3,5-Difluorophenacetyl))-L-alanyl)-S-phenylglycerin t-butyl ester. By introducing these targeted conjugated nanoparticles, detection, targeting, and destruction of the Hepatocarcinoma and glioblastoma stem cells was achieved. An efficient alternative treatment for the incurable disease of cancer could be provided.Article history: This study was aimed to evaluate the in vitro complexation nature and strength of complex which may be formed due to interaction between Amoxicillin and Calcium chloride (CaCl2). The interaction of Amoxicillin and Calcium chloride (fused) has been studied in aqueous systems at a fixed temperature (37 ± 0.5) °C and under different pH (pH 2.4 and pH 7.4) by using some physical methods as spectral observation, Jobs method of continuous variation, Ardons method. From spectrophotometric study, Amoxicillin gives a sharp peak at 272 nm when Calcium chloride mixed with Amoxicillin in 1:1 ratio the intensity of the peak of Amoxicillin change remarkably due to interaction. The jobs plot was obtained by plotting absorbance difference against the mole fraction of the each drug at pH 2.4 and pH 7.4. Amoxicillin forms strong 1:1 complex with Calcium chloride and reverse V Shaped curves indicate the formation of 1:1 complexes of Amoxicillin with Calcium chloride. These may indicate strong kinetics of complexation between Amoxicillin with Calcium chloride. The value of stability constant for the complexation of Amoxicillin with calcium chloride at pH 2.4 and pH 7.4 were obtained from the spectral data using Ardons plot. The value of stability constant for the drug-metal system at pH 2.4 and pH 7.4 are 5.54 and 6.67 respectively. At pH 2.4 it is found that Amoxicillin form relatively stable complex with Calcium chloride (stability constant 6.67) is high in comparison to pH 7.4. It can therefore be concluded that a careful consideration is needed during concurrent administration of Amoxicillin with Calcium chloride.A deficit/hyperactivity disorder (ADHD) is among the most frequent neurodevelopmental disorders. Atomoxetine (ATX) and methylphenidate (MPH) have been recommended as primary medication choices to treat inhibitionand attention-related dysfunctions in ADHD children. This study used functional near-infrared spectroscopy (fNIRS) to explore the efficacy of both medications in school-aged children with ADHD for inhibitory and attention task performance. fNIRS is a promising tool, offering robust advantages such as its compactness, affordable price, tolerance to body motion and accessibility. We monitored the oxy-hemoglobin changes in ADHD children (6 to 14 years old) during go/nogo or oddball tasks before and 1.5 h after ATX, MPH or placebo administration, in a randomized, double-blind, placebo-controlled experiment. Age-, genderand IQ-matched healthy controls, who did not receive medications or a placebo, were also monitored. In the control subjects, the go/nogo task modulated the right inferior and middle prefrontal gyri (IFG/MFG) and the oddball task modulated the right IFG/MFG and inferior parietal cortex (IPL). In ADHD children, these activations were absent in pre-medicated conditions. The reduction in the right IFG/MFG activation was normalized by both ATX and MPH for go/nogo and oddball tasks, but the right IPL was normalized only by ATX in the oddball task. These results led us to conclude that fNIRS could visualize the differential neuropharmacological effects of both substances in the inhibitory and attentional networks: ATX to up-regulate the noradrenergic system reflected in the right IFG/MFG and IPL activations, and MPH to up-regulate the dopamine system reflected in the IFG/MFG activations.G a bioactive compound found in many Traditional Chinese medicine and Ayurvedic herbs. It was recently shown to be in abundance in the rind waste of the fruit Nephelium lappaceum. We have shown in our laboratories, using both in vitro and in vivo studies, the ability of this compound extracted from Nephelium lappaceum to effectively enhance glucose uptake, reduce insulin resistance, reduce obesity among other uses. Others have shown the anti-hypertensive ability of the compound.