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Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes

Abd Ghafar N, Chua KH, Wan Ngah WZ, Che Hamzah J, Othman F, Abd Rahman R, et al.

Cell Tissue Bank, 2014 Mar;15(1):25-34.
PMID: 23292197 DOI: 10.1007/s10561-012-9360-y

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.

Matched MeSH terms: Actins/biosynthesis
Fulltext Identification of AOC3 and LRRC17 as Colonic Fibroblast Activation Markers and Their Potential Roles in Colorectal Cancer Progression

Ahmad Zawawi SS, Mohd Azram NAS, Sulong S, Zakaria AD, Lee YY, Che Jalil NA, et al.

Asian Pac J Cancer Prev, 2023 Sep 01;24(9):3099-3107.
PMID: 37774061 DOI: 10.31557/APJCP.2023.24.9.3099

BACKGROUND: Accumulation of cancer-associated fibroblasts (CAFs) in the tumor stroma is linked to poor prognosis in colorectal cancer (CRC). CAF-cancer cell interplay, facilitated by secretomes including transforming growth factor-beta 1 (TGF-β1), supports fibroblast activation, drives colorectal carcinogenesis, and contributes to CRC aggressive phenotypes. Although widely used, traditional CAF biomarkers are found to have heterogeneous and non-specific expression. Amine oxidase copper containing 3 (AOC3) and leucine-rich repeat-containing 17 (LRRC17) have been reported to be emerging markers of myofibroblasts.
AIM: Our objective was to investigate the potential of AOC3 and LRRC17 as biomarkers for fibroblast activation thus predicting their roles in CRC progression.
METHODS: Immunofluorescence (IF) staining of AOC3 and LRRC17 was performed on myofibroblast line (CCD-112CoN), primary fibroblasts from colorectal tumor (CAFs), and adjacent normal tissue (normal fibroblasts-NFs). SW620 (epithelial CRC cell line) was used as a control. Conventional CAF biomarker (alpha-smooth muscle actin - α-SMA) was included in the IF analysis. Fluorescence intensity was compared between groups using ImageJ software. Proliferation and contractility of treated cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and collagen gel contraction assays, respectively. Fibroblast contraction under TGF-β1 treatment was compared to those treated with complete medium (addition of 10% serum) and serum free (SF) medium.
RESULTS: Positive AOC3, LRRC17, and α-SMA expression were observed in colonic fibroblasts, more prominent in CAFs, whereas negative staining was found in SW620. Significant downregulation of AOC3, and upregulations in LRRC17 and α-SMA expression was found in TGF-β1-treated fibroblasts compared to SF medium treatment (p-value<0.05). All fibroblasts exhibited higher proliferation in complete medium and under treatment with conditioned medium from SW620 than SF medium. Significant contraction of NFs was recorded in complete medium and TGF-β1 (p-value<0.01).
CONCLUSION: Our results demonstrate AOC3 and LRRC17 as the potential markers of CAF activation which promote CRC progression.

Matched MeSH terms: Actins/metabolism
Fulltext Congenital epulis: immunohistochemical findings of 12 cases

Ajura Abdul Jalil, Lukman Md Auzair, Hin, Lau Shin

Archives of Orofacial Sciences, 2015;10(1):34-40.
MyJurnal

Congenital epulis is a fairly rare soft tissue tumour occurring exclusively on the alveolar ridge of newborns. The exact origin of congenital epulis is still debatable. The objective of the study is to determine the clinicopathological features and immunohistochemical findings of congenital epulis. A retrospective study was carried out to determine the clinicopathological features of congenital epulis, diagnosed histologically in the main oral histopathology laboratory in Malaysia from 1967 to 2014. Immunostaining using vimentin, muscle specific actin, smooth muscle antigen, desmin, S100, CD34, CD68 and CD1a was carried out. Twelve cases of congenital epulis were reviewed. All of the patients were females and the presentation age ranged from 2 to 90 days. The patients comprised of 6 Malays, 3 Chinese, 2 Indians and 1 Orang Asli. Most of the cases (n=7) involved the maxillary ridge and presented as pedunculated well-defined lumps (n=8). Excisional biopsy was performed in all cases. Via immunohistochemistry, vimentin expression was observed in all cases; but negative for CD34, muscle specific actin, smooth muscle antigen, and desmin. CD1a and S100 positivity was seen in five cases. The interstitial cells were highlighted by CD68. Although congenital epulis has been first described 130 years ago, the exact nature of its histogenesis remains a mystery.

Matched MeSH terms: Actins
Improvement of early cell adhesion on Thai silk fibroin surface by low energy plasma

Amornsudthiwat P, Mongkolnavin R, Kanokpanont S, Panpranot J, Wong CS, Damrongsakkul S

Colloids Surf B Biointerfaces, 2013 Nov 1;111:579-86.
PMID: 23893032 DOI: 10.1016/j.colsurfb.2013.07.009

Low energy plasma has been introduced to treat the surface of Thai silk fibroin which should be enhanced for cell adhesion due to its native hydrophobic surface. Plasma surface treatment could introduce desirable hydrophilic functionalities on the surface without using any chemicals. In this work, nitrogen glow discharge plasma was generated by a low energy AC50Hz power supply system. The plasma operating conditions were optimized to reach the highest nitrogen active species by using optical emission spectroscopy. X-ray photoelectron spectroscopy (XPS) revealed that amine, hydroxyl, ether, and carboxyl groups were induced on Thai silk fibroin surface after plasma treatment. The results on Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy confirmed that the plasma treated effects were only on the outermost layer since there was no change in the bulk chemistry. The surface topography was insignificantly changed from the detection with atomic force microscopy (AFM). The plasma-treated effects were the improved surface wettability and cell adhesion. After a 90-s treatment, the water contact angle was at 20°, while the untreated surface was at 70°. The early cell adhesion of L929 mouse fibroblast was accelerated. L929 cells only took 3h to reach 100% cell adhesion on 90 s N2 plasma-treated surface, while there was less than 50% cell adhesion on the untreated Thai silk fibroin surface after 6h of culture. The cell adhesion results were in agreement with the cytoskeleton development. L929 F-actin was more evident on 90 s N2 plasma-treated surface than others. It could be concluded that a lower energy AC50Hz plasma system enhanced early L929 mouse fibroblast adhesion on Thai silk fibroin surface without any significant change in surface topography and bulk chemistry.

Matched MeSH terms: Actins/metabolism
An ultrastructural study on the Leydig and Sertoli cells in the immature lesser mouse deer (Tragulus javanicus)

Andriana BB, Kanai Y, Kimura J, Fukuta K, Hayashi Y, Kurohmaru M

Anat Histol Embryol, 2005 Jun;34(3):171-5.
PMID: 15929732

Leydig and Sertoli cells of the immature lesser mouse deer testes, obtained in East Malaysia, were observed using light and transmission electron microscopy (TEM). The testes were fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethanol, and embedded in Araldite M. Serial semi-thin sections were cut, stained with toluidine blue and observed using light microscopy. Serial ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and examined using TEM. As a result, ultrastructurally, two types of underdeveloped filament bundles were infrequently recognized in Leydig cells, but not in other testicular cells. One type was the underdeveloped bundles of actin filaments (approximately 5 nm in diameter), which were found in the nucleus of Leydig cells. The other type was the underdeveloped bundles of intermediate filaments (approximately 10 nm in diameter), which were found in the cytoplasm of Leydig cells. A multivesicular nuclear body (MNB)--specifically present in the Sertoli cell nucleus of ruminant testes--was infrequently observed. The MNB is situated in the vicinity of nuclear membrane, still in an underdeveloped stage.

Matched MeSH terms: Actins/ultrastructure
Fulltext Attachment, Proliferation, and Morphological Properties of Human Dermal Fibroblasts on Ovine Tendon Collagen Scaffolds: A Comparative Study

Busra FM, Lokanathan Y, Nadzir MM, Saim A, Idrus RBH, Chowdhury SR

Malays J Med Sci, 2017 Mar;24(2):33-43.
PMID: 28894402 DOI: 10.21315/mjms2017.24.2.5

INTRODUCTION: Collagen type I is widely used as a biomaterial for tissue-engineered substitutes. This study aimed to fabricate different three-dimensional (3D) scaffolds using ovine tendon collagen type I (OTC-I), and compare the attachment, proliferation and morphological features of human dermal fibroblasts (HDF) on the scaffolds.
METHODS: This study was conducted between the years 2014 to 2016 at the Tissue Engineering Centre, UKM Medical Centre. OTC-I was extracted from ovine tendon, and fabricated into 3D scaffolds in the form of sponge, hydrogel and film. A polystyrene surface coated with OTC-I was used as the 2D culture condition. Genipin was used to crosslink the OTC-I. A non-coated polystyrene surface was used as a control. The mechanical strength of OTC-I scaffolds was evaluated. Attachment, proliferation and morphological features of HDF were assessed and compared between conditions.
RESULTS: The mechanical strength of OTC-I sponge was significantly higher than that of the other scaffolds. OTC-I scaffolds and the coated surface significantly enhanced HDF attachment and proliferation compared to the control, but no differences were observed between the scaffolds and coated surface. In contrast, the morphological features of HDF including spreading, filopodia, lamellipodia and actin cytoskeletal formation differed between conditions.
CONCLUSION: OTC-I can be moulded into various scaffolds that are biocompatible and thus could be suitable as scaffolds for developing tissue substitutes for clinical applications and in vitro tissue models. However, further study is required to determine the effect of morphological properties on the functional and molecular properties of HDF.

Matched MeSH terms: Actins
The role of host microfilaments and microtubules during opsonin-independent interactions of Cryptococcus neoformans with mammalian lung cells

Choo KK, Chong PP, Ho AS, Yong PV

Eur J Clin Microbiol Infect Dis, 2015 Dec;34(12):2421-7.
PMID: 26463450 DOI: 10.1007/s10096-015-2497-4

The purpose of this investigation was to characterise the interactions of Cryptococcus neoformans with mammalian host alveolar epithelial cells and alveolar macrophages, with emphasis on the roles of the cryptococcal capsule and the host cell cytoskeletons. The adherence and internalisation of C. neoformans into mammalian lung cells and the roles of host cell cytoskeletons in host-pathogen interactions were studied using in vitro models coupled with a differential fluorescence assay, fluorescence staining, immunofluorescence and drug inhibition of actin and microtubule polymerisation. Under conditions devoid of opsonin and macrophage activation, C. neoformans has a high affinity towards MH-S alveolar macrophages, yet associated poorly to A549 alveolar epithelial cells. Acapsular C. neoformans adhered to and internalised into the mammalian cells more effectively compared to encapsulated cryptococci. Acapsular C. neoformans induced prominent actin reorganisation at the host-pathogen interface in MH-S alveolar macrophages, but minimally affected actin reorganisation in A549 alveolar epithelial cells. Acapsular C. neoformans also induced localisation of microtubules to internalised cryptococci in MH-S cells. Drug inhibition of actin and microtubule polymerisation both reduced the association of acapsular C. neoformans to alveolar macrophages. The current study visualises and confirms the interactions of C. neoformans with mammalian alveolar cells during the establishment of infection in the lungs. The acapsular form of C. neoformans effectively adhered to and internalised into alveolar macrophages by inducing localised actin reorganisation, relying on the host's actin and microtubule activities.

Matched MeSH terms: Actins
Fulltext Cell surface proteins of cryptococcus neoformans mediate adherence and internalisation into mammalian cells

Choo, K.K., Chin, V.K., Chong, P.P., Ho, S.H., Yong, V.C.

JUMMEC, 2019;22(2):24-30.
MyJurnal

Cryptococcus neoformans is an encapsulated fungal pathogen that causes severe disease primarily in
immunocompromised patients. Adherence and internalisation of microbial pathogens into host cells often
begin with engagement of microbes to the surface receptors of host. However, the mechanisms involved
remain poorly understood. In this study, we investigated the association of cell surface determinants of C.
neoformans with mammalian cells. Our results showed that treatment with trypsin, but not paraformaldehyde
or heat killing, could reduce host-cryptococci interaction, suggesting the involvement of cell surface proteins
(CSPs) of C. neoformans in the interaction. We extended our investigations to determine the roles of CSPs
during cryptococci-host cells interaction by extracting and conjugating CSPs of C. neoformans to latex beads.
Conjugation of CSPs with both encapsulated and acapsular C. neoformans increased the association of latex
beads with mammalian alveolar epithelial cells, alveolar macrophages and monocyte-derived macrophages.
Further examination on the actin organisation of the host cells implied the involvement of actin-dependent
phagocytosis in the internalisation of C. neoformans in CSP-conjugated latex beads. We hypothesised that
CSPs present on the cell wall of C. neoformans mediate the adherence and actin-dependent phagocytosis
of cryptococci by mammalian cells. Our results warrant further studies on the exact role of CSPs in the
pathogenesis of cryptococcosis.

Matched MeSH terms: Actins
Fulltext Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos

Dasiman R, Rahman NS, Othman S, Mustafa MF, Yusoff NJ, Jusoff WH, et al.

Med Sci Monit Basic Res, 2013 Oct 04;19:258-66.
PMID: 24092420 DOI: 10.12659/MSMBR.884019

BACKGROUND: This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM).
MATERIAL/METHODS: Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.
RESULTS: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.
CONCLUSIONS: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.

Matched MeSH terms: Actins/metabolism
Fulltext The Cytoplasmic Actins in the Regulation of Endothelial Cell Function

Dugina VB, Shagieva GS, Shakhov AS, Alieva IB

Int J Mol Sci, 2021 Jul 22;22(15).
PMID: 34360602 DOI: 10.3390/ijms22157836

The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate permeability of vascular walls and to control exchange between circulating blood and tissue fluids of organs. The EC actin cytoskeleton plays a crucial role in maintaining endothelial barrier function. Actin cytoskeleton reorganization result in EC contraction and provides a structural basis for the increase in vascular permeability, which is typical for many diseases. Actin cytoskeleton in non-muscle cells presented two actin isoforms: non-muscle β-cytoplasmic and γ-cytoplasmic actins (β-actins and γ-actins), which are encoded by ACTB and ACTG1 genes, respectively. They are ubiquitously expressed in the different cells in vivo and in vitro and the β/γ-actin ratio depends on the cell type. Both cytoplasmic actins are essential for cell survival, but they perform various functions in the interphase and cell division and play different roles in neoplastic transformation. In this review, we briefly summarize the research results of recent years and consider the features of the cytoplasmic actins: The spatial organization in close connection with their functional activity in different cell types by focusing on endothelial cells.

Matched MeSH terms: Actins/metabolism*
Gene expression characteristic in human auricular cartilage tissue engineering

Farah Wahida I, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Isa MR, et al.

Med J Malaysia, 2004 May;59 Suppl B:190-1.
PMID: 15468882

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.

Matched MeSH terms: Actins/genetics
Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells

Fong LY, Ng CT, Zakaria ZA, Baharuldin MT, Arifah AK, Hakim MN, et al.

Phytother Res, 2015 Oct;29(10):1501-8.
PMID: 26171791 DOI: 10.1002/ptr.5404

The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

Matched MeSH terms: Actins
Asiatic acid stabilizes cytoskeletal proteins and prevents TNF-α-induced disorganization of cell-cell junctions in human aortic endothelial cells

Fong LY, Ng CT, Yong YK, Hakim MN, Ahmad Z

Vascul. Pharmacol., 2019 06;117:15-26.
PMID: 30114509 DOI: 10.1016/j.vph.2018.08.005

Endothelial hyperpermeability represents an initiating step in early atherosclerosis and it often occurs as a result of endothelial barrier dysfunction. Asiatic acid, a major triterpene isolated from Centella asiatica (L.) Urban, has previously been demonstrated to protect against tumor necrosis factor (TNF)-α-induced endothelial barrier dysfunction. The present study aimed to investigate the mechanisms underlying the barrier protective effect of asiatic acid in human aortic endothelial cells (HAECs). The localization of F-actin, diphosphorylated myosin light chain (diphospho-MLC), adherens junctions (AJs) and tight junctions (TJs) was studied using immunocytochemistry techniques and confocal microscopy. Their total protein expressions were examined using western blot analysis. The endothelial permeability was assessed using In Vitro Vascular Permeability Assay kits. In addition, intracellular redistribution of the junctional proteins was evaluated using subcellular fractionation kits. We show that asiatic acid stabilized F-actin and diphospho-MLC at the cell periphery and prevented their rearrangement stimulated by TNF-α. However, asiatic acid failed to attenuate cytochalasin D-induced increased permeability. Besides, asiatic acid abrogated TNF-α-induced structural reorganization of vascular endothelial (VE)-cadherin and β-catenin by preserving their reticulum structures at cell-cell contact areas. In addition, asiatic acid also inhibited TNF-α-induced redistribution of occludin and zona occludens (ZO)-1 in different subcellular fractions. In conclusion, the barrier-stabilizing effect of asiatic acid might be associated with preservation of AJs and prevention of TJ redistribution caused by TNF-α. This study provides evidence to support the potential use of asiatic acid in the prevention of early atherosclerosis, which is initiated by endothelial barrier dysfunction.

Matched MeSH terms: Actins/metabolism
Fulltext Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

Gholami K, Loh SY, Salleh N, Lam SK, Hoe SZ

PLoS One, 2017;12(6):e0176368.
PMID: 28591185 DOI: 10.1371/journal.pone.0176368

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Matched MeSH terms: Actins/biosynthesis
Fulltext Invadopodia formation is a critical step in cancer cell invasion: the effect of passage number on invadopodia formation in MDA-MB-231 breast cancer cell line

Hamad Ali Hamad, Cheah, Yoke Kqueen, Nur Fariesha MD Hashim

Life Sciences, Medicine and Biomedicine, 2018;2(2):1-5.
MyJurnal

High invasive cancer cells are thought to recruit specialised actin-rich protrusions for invasion in metastasis process. These protrusions are termed invadopodia. To study invadopodia formation, one of the first challenges faced by researchers has been to optimise the cell line passage number in order to be used for the invadopodia assay. Therefore, this study aims to investigate the effects of the passage number on invadopodia formation in MDA-MB-231 breast cancer cell line. Invadopodia assay was used to achieve the aim of the study. The results provided evidence that invadopodia formation is affected by the high passage number. The cells were also tested with dimethyloxalylglycine (DMOG) a hypoxic mimicking agent which is known to be an invadopodia inducer, the results showed that the cells in low passage number (P7) treated with DMOG increase the cells forming invadopodia, while the cells with high passage number (P35) showed that DMOG fails to stimulate the cells to form invadopodia. Furthermore, the cells with high passage number after passage 15 are starting to lose the ability to degrade the gelatin. In conclusion, this study suggests that only cells with a low passage number, less than passage 15 should be used in the study of invadopodia formation to obtain the results in the search for molecular targets and signaling at invadopodia.

Matched MeSH terms: Actins
Effects of Nicotine and Tocotrienol-Rich Fraction Supplementation on Cytoskeletal Structures of Murine Pre-Implantation Embryos

Hamirah NK, Kamsani YS, Mohamed Nor Khan NA, Ab Rahim S, Rajikin MH

Med Sci Monit Basic Res, 2017 Dec 08;23:373-379.
PMID: 29217815

BACKGROUND Cytoskeletal structures, in particular actin and tubulin, provide a fundamental framework in all cells, including embryos. The objective of this study was to evaluate the effects of nicotine, which is a source of oxidative stress, and subsequent supplementation with Tocotrienol-rich fraction (TRF) on actin and tubulin of 2- and 8-cell murine embryos. MATERIAL AND METHODS Thirty female Balb/C mice were divided into 4 groups: Group 1 received: subcutaneous (sc) injection of 0.9% NaCl; Group 2 received sc injection of 3.0 nicotine mg/kg bw/day; Group 3 received 3.0 sc injection of nicotine mg/kg bw/day +60 mg/kg bw/day TRF; and Group 4 received 60 sc injection of TRF mg/kg bw/day for 7 consecutive days. The animals were superovulated with 5 IU PMSG followed by 5 IU hCG 48 h later. Animals were cohabited with fertile males overnight and euthanized through cervical dislocation at 24 h post coitum. Embryos at the 2- and 8-cell stages were harvested, fixed, and stained to visualize actin and tubulin distributions by using CLSM. RESULTS Results showed that at 2-cell stage, actin intensities were significantly reduced in the nicotine group compared to that of the control group (p<0.001). In Group 3, the intensity of actin significantly increased compared to that of the nicotine group (p<0.001). At 8-cell stage, actin intensity of the nicotine group was significantly lower than that of the control group (p<0.001). The intensities of actin in Group 3 were increased compared to that of nicotine treatment alone (p<0.001). The same trend was seen in tubulin at 2- and 8-cell stages. Interestingly, both actin and tubulin structures in the TRF-treated groups were enhanced compared to the control. CONCLUSIONS This study suggests that TRF prevents the deleterious effects of nicotine on the cytoskeletal structures of 2- and 8-cell stages of pre-implantation mice embryos in vitro.

Matched MeSH terms: Actins/drug effects
Fulltext Data on PCR primer design for glucose 6-phosphate dehydrogenase gene and the effects of dietary carbohydrate levels on its expression in the liver of Malaysian mahseer (Tor tambroides)

Ishak SD, Razali SA, Kamarudin MS, Abol-Munafi AB

Data Brief, 2020 Aug;31:105916.
PMID: 32642522 DOI: 10.1016/j.dib.2020.105916

The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyses the metabolite glucose-6-phosphate in producing NADPH during the first phase of pentose-phosphate pathway thus provides reducing power to all cells for cellular growth, antioxidant defence, and biosynthetic reactions in all living organism. The deliberate inclusion of starch as carbohydrate source in commercial feed however may affect the G6PD hepatic activity in cultured fish. We designed a set of primers to target G6PD gene in the popular Malaysian aquaculture species, Tor tambroides. For this dataset, the molecular characteristics of obtained T. tambroides G6PD (TtG6PD) nucleotide sequence was analysed using multiple alignments and phylogenetic analyses of the deduced amino acids. The set of primers obtained were then used in a study to evaluate the effect of different dietary carbohydrate inclusion levels on the hepatic TtG6PD mRNA expression of the T. tambroides fingerlings. Four groups of fish were given a dietary treatment of 15%, 20%, 25% and 30% starch at the optimal inclusion level of 23.4% for 10 weeks. The TtG6PD mRNA transcripts were measured using real-time-PCR assays and its expression normalized against β-actin, which acts as the internal control gene. This article provides supportive data in relation between hepatic TtG6PD mRNA gene expression in T. tambroides and how it is influenced by its dietary carbohydrate intake. These data will also assist in further nutritional genomic studies of carbohydrate and energy utilization for all species in the mahseer family.

Matched MeSH terms: Actins
Identification of Common and Novel Major Crab Allergens in Scylla tranquebarica and the Allergen Stability in Untreated and Vinegar-treated Crab

Jasim HA, Misnan R, Yadzir ZHM, Abdullah N, Bakhtiar F, Arip M, et al.

Iran J Allergy Asthma Immunol, 2021 Feb 11;20(1):76-87.
PMID: 33639634 DOI: 10.18502/ijaai.v20i1.5414

Crab allergy is reported as a serious form of food allergy in many countries. This study was aimed to identify the major allergens of the local mud crab, Scylla tranquebarica (S. tranquebarica), and subsequently, determine the effect of vinegar treatments on the crab allergens. Crab muscles were treated with synthetic and natural vinegar. Crab proteins were then extracted from the untreated and vinegar-treated crabs. All extracts were then fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by immunoblotting; using sera from crab-allergic patients. The crab proteins were then further fractionated by two-dimensional electrophoresis (2-DE)and analyzed by mass spectrometry (MS). The untreated crab had 38 protein bands, while that was only a few bands between 18 to 73 kDa for the vinegar-treated crabs. Immunoblotting of untreated crab revealed 20 IgE-binding bands, whereas the vinegar-treated crabs could only retain a few IgE-binding bands. Five major allergens were identified with molecular weightsof38, 42, 49, 63, and 73 kDa in the untreated crab. In contrast, the vinegar-treated crabs had only a few major allergens with molecular weights of 38, 42, and 73 kDa. MS identified the 43 and 49 kDa as arginine kinase, while the 38, 63, and 73 kDa were identified as tropomyosin, actin, and hemocyanin, respectively. Inconclusion, we found three common major allergens for S. tranquebarica including tropomyosin, arginine kinase, and actin, and one novel allergen known as hemocyanin. All the major allergens could retain minimal allergenic capability in vinegar-treated crabs, suggesting that vinegar treatments might be useful to reduce crab allergenicity. These data would assist the clinicians in the management of crab-allergic patients worldwide.

Matched MeSH terms: Actins
Fulltext Protective effects of citrullus vulgaris on irradiated lymphocyte submembrane

Khairul Osman, Norashikin Md.Saad, Ezlan Elias, Siti Fatimah Ibrahim, Jamaludin Mohamed, Proomwichit, Proom

Jurnal Sains Kesihatan Malaysia, 2005;3(1):1-9.
MyJurnal

A study was conducted to determine the radioprotective effects of Citrullus vulgaris on the lymphocyte sub-membrane particularly the actin layer. A total of 30 adult male Sprague-Dawley rats were divided into three equal groups of positive control, negative control and treatment. The positive and negative control groups were force fed with 40 ml/kg body weight of normal saline while the treatment group received 40 g/kg body weight of fresh juice of C. vulgaris daily. After a week the positive control and treatment groups were irradiated with 90 rad gamma radiation. Viable lymphocytes were determined using propidium iodine and acridine orange stain and observed under a fluorescent microscope. The percentage of viable lymphocytes of the treatment group (71.0%; p = 0.03) was significantly higher than the positive control group. The results showed that C. vulgaris possessed radioprotective effects because the lymphocyte actin was not damaged. The radioprotection effects could be due to the presence of antioxidants in C. vulgaris.

Matched MeSH terms: Actins
Fulltext A clinical and immunohistochemical study of gastrointestinal stromal tumours

Khoo JJ, Gunn A

Malays J Pathol, 2005 Jun;27(1):9-16.
PMID: 16676687

AIM: To study the clinical features, histology and immunohistochemical properties of gastrointestinal stromal tumours (GISTs); and establish any parameters that can help prognosticate the malignant potential.
METHODS: Twenty-six patients with GISTs who were seen in Sultanah Aminah Hospital Johor, Malaysia from 1999 to 2003 were selected for study. Patient, clinical characteristics and outcome based on surgical records were analysed. Tumour variables (tumour size, cellularity, mitotic count, necrosis and haemorrhage) were compared between very low to low risk groups and intermediate to high risk groups. The immunohistochemical properties of GISTs were also studied.
RESULTS: Patients with GISTs presented mainly with pain, palpable mass or gastrointestinal tract bleeding. The tumours were seen in stomach (50%) followed by small intestine (38.5%) and rectum (11.5%). In the period of study, six patients had metastasis, mainly in the liver or peritoneum. Immunoreactivity for CD117, CD34, vimentin, S100, neuron specific enolase, alpha-smooth-muscle-actin and desmin were observed in 100%, 76.9%, 61.5%, 46.1%, 80.8%, 11.5% and 0% of tumours respectively. The behaviour of GISTs was largely dependent on tumour size and number of mitosis. Necrosis and haemorrhage were seen in tumours with high risk potential.

Matched MeSH terms: Actins/immunology; Actins/metabolism

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