The disease Tuberculosis (TB) is caused by a bacterium called Mycobacterium tuberculosis (Mtb). The bacterial cell-wall consists of peptidoglycan layer maintains the cellular integrity and cell viability. The main problem resides in the cell cycle of Mycobacterium tuberculosis in its quiescent form which is not targeted by any drugs hence there is an immediate need for new antibiotics to target the cell wall. The current study deals with the dTDP-4-dehydrorahmnose reductase (RmlD) which is the final enzyme in the series of cell-wall proteins of Mtb. The RmlD is a part of Carbohydrate biosynthesis has been considered as a good drug target for the novel class of antibiotics. Our study begins with the protein structure prediction, Homology studies were conducted using the Phyre2 web server. The structure is then refined and subjected to molecular dynamics simulations for 50 ns using GROMACS. The clustering analysis has been carried out and generated 41 clusters with 2 Å as the cut-off. Blind docking virtual screening was performed against RmlD protein using the Super Natural-II database with AutoDock4.0. its results helped to screen top ligands based on best binding energies. In both dockings, there are some common residues in which the ligands are interacting and forming the Hydrogen bonds such as Asp-105, Val-158, Thr-160, Gly-161, Arg-224, Arg-256. The ligand-567 giving the best results by being in the top-3 of all the clusters in both blind docking as well as the active-site docking. Hence ligand-567 can be a potential inhibitor of RmlD which can further inhibit the cell-wall synthesis of Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.
In-stent restenosis is caused by the proliferation of the smooth muscle cells (SMCs) following a host response towards the implanted device. However, the precise biochemical and cellular mechanisms are still not completely understood. In this paper, the behaviour of SMCs has been investigated by an in vitro model where the cells were stimulated by platelet derived growth factor (PDGF) on tissue-like substrates as well as on biomaterials such as stainless steel (St) and diamond-like carbon (DLC)-coated St. The results demonstrated that SMCs have a completely different adhesion mode on St and become particularly prone to proliferation and pro-inflammatory cytokine secretion under PDGF stimulus. This would suggest that restenosis may caused by the accidental contact of the SMC with the St substrate under an inflammatory insult.
Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated. One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12. This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.
Biocompatibility of two variants of accelerated Portland cement (APC) were investigated in vitro by observing the cytomorphology of SaOS-2 osteosarcoma cells in the presence of test materials and the effect of these materials on the expression of markers of bone remodelling. Glass ionomer cement (GIC), mineral trioxide aggregate (MTA) and unmodified Portland cement (RC) were used for comparison. A direct contact assay was undertaken in four samples of each test material, collected at 12, 24, 48 and 72 h. Cell morphology was observed using scanning electron microscopy (SEM) and scored. Culture media were collected for cytokine quantification using enzyme-linked immunosorbent assay (ELISA). On SEM evaluation, healthy SaOS-2 cells were found adhering onto the surfaces of APC variant, RC and MTA. In contrast, rounded and dying cells were observed on GIC. Using ELISA, levels of interleukin (IL)-1beta, IL-6, IL-18 and OC were significantly higher in APC variants compared with controls and GIC (p<0.01), but these levels of cytokines were not statistically significant compared with MTA. The results of this study provide evidence that both APC variants are non-toxic and may have potential to promote bone healing. Further development of APC is indicated to produce a viable dental restorative material and possibly a material for orthopaedic
Camel milk has been gaining immmense importance due to high nutritious value and medicinal properties. Peptides from milk proteins is gaining popularity in various therapeutics including human cancer. The study was aimed to investigate the anti-cancerous and anti-inflammatory properties of camel whey protein hydrolysates (CWPHs). CWPHs were generated at three temperatures (30 ℃, 37 ℃, and 45 ℃), two hydrolysis timepoints (120 and 360 min) and with three different enzyme concentrations (0.5, 1 and 2 %). CWPHs demonstrated an increase in anti-inflammatory effect between 732.50 (P-6.1) and 3779.16 (P-2.1) µg Dicolfenac Sodium Equivalent (DSE)/mg protein. CWPHs (P-4.3 & 5.2) inhibited growth of human colon carcinoma cells (HCT116) with an IC50 value of 231 and 221 μg/ml, respectively. P-4.3 induced G2/M cell cycle arrest and modulated the expression of Cdk1, p-Cdk1, Cyclin B1, p-histone H3, p21 and p53. Docking of two peptides (AHLEQVLLR and ALPNIDPPTVER) from CWPHs (P-4.3) identified Polo like kinase 1 as a potential target, which strongly supports our in vitro data and provides an encouraging insight into developing a novel peptide-based anticancer formulation. These results suggest that the active component, CWPHs (P-4.3), can be further studied and modeled to form a small molecule anti-cancerous therapy.
In this study, the effects of Chlorella vulgaris (CV) on replicative senescence of human diploid fibroblasts (HDFs) were investigated. Hot water extract of CV was used to treat HDFs at passages 6, 15, and 30 which represent young, presenescence, and senescence ages, respectively. The level of DNA damage was determined by comet assay while apoptosis and cell cycle profile were determined using FACSCalibur flow cytometer. Our results showed direct correlation between increased levels of damaged DNA and apoptosis with senescence in untreated HDFs (P < 0.05). Cell cycle profile showed increased population of untreated senescent cells that enter G0/G1 phase while the cell population in S phase decreased significantly (P < 0.05). Treatment with CV however caused a significant reduction in the level of damaged DNA and apoptosis in all age groups of HDFs (P < 0.05). Cell cycle analysis showed that treatment with CV increased significantly the percentage of senescent HDFs in S phase and G2/M phases but decreased the population of cells in G0/G1 phase (P < 0.05). In conclusion, hot water extract of Chlorella vulgaris effectively decreased the biomarkers of ageing, indicating its potential as an antiageing compound.
Six Malaysian chloroquine-resistant Plasmodium falciparum isolates were cultured in vitro following the candle-jar method. Antimalarial evaluations of daily replacement of culture medium containing chloroquine and a semi-purified extract of Eurycoma longifolia Jack (containing 13 beta, 18-dihydroeurycomanol (1), eurycomanol-2-O-beta-D-glucopyranoside (2), eurycomanol (3) and eurycomanone (4)) were performed on 6-well plates at 37 degrees C for a week. Presence or absence of the parasites was determined microscopically on thin-film Giemsa-stained preparations. Results showed that the antimalarial activity of Eurycoma longifolia Jack was dose-dependent and reached a maximum of < 50% at 0.07-5.00 micrograms ml-1 after 1 day post-treatment. However, complete inhibitions were observed at 1.25-5.00 micrograms ml-1 extract after 3 days post-treatment and 0.62 and 0.31 micrograms ml-1 after 4 and 6 days post-treatment, respectively. Further results indicated that chloroquine exhibited total inhibition at concentrations > 2.50 and 0.62 micrograms ml-1 after 1 and 2 days post-treatment, respectively and at all concentrations after 3 days post-treatment.
Leea indica is a medicinal plant used traditionally to cure cancer. In this study, the cytotoxic compounds of L. indica were isolated using bioassay-guided approach. Two cycloartane triterpenoid glycosides, mollic acid arabinoside (MAA) and mollic acid xyloside (MAX), were firstly isolated from L. indica. They inhibited the growth of Ca Ski cervical cancer cells with IC(50) of 19.21 μM (MAA) and 33.33 μM (MAX). MRC5 normal cell line was used to calculate selectivity index. MAA and MAX were about 8 and 4 times more cytotoxic to Ca Ski cells compared to MRC5. The cytotoxicity of MAA was characterized by both cytostatic and cytocidal effects. MAA decreased the expression of proliferative cell nuclear antigen, increased sub-G1 cells, and arrested cells in S and G2/M phases. This study provides the evidence for the ethnomedicinal use of L. indica and paves the way for future mechanism studies on the anticancer effects of MAA.
Transforming growth factor-beta (TGF-beta) has been shown to inhibit the growth of mammary epithelial cells and may play a protective role in mammary carcinogenesis. In contrast, oestrogens promote the development of breast cancer. Oestrone sulphate (E1S) is a huge reservoir of active oestrogens in the breast being converted to the weak oestrogen, oestrone (E1), by oestrone sulphatase. E1 is reversibly converted by oestradiol-17beta hydroxysteroid dehydrogenase to the potent oestrogen, oestradiol (E2). The aim of this study was to assess the effect of the TGF-beta1 isoform on growth and oestrogen metabolism in the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 human breast cancer cell lines. The results showed that TGF-beta1 significantly inhibited cell growth and stimulated the conversion of E1S to E1 and E1 to E2 in the MCF-7 cell line. In the MDA-MB-231 cell line TGF-beta1 significantly stimulated cell growth and inhibited the interconversions between E1 and E2. In conclusion, the growth inhibitory effect of TGF-beta1 on the MCF-7 cell line would appear to confer a protective effect in breast cancer. However, its ability to increase the amount of E2 would increase the risk of breast cancer. Which of these effects predominates in vivo remains to be explored. The growth stimulatory effect of TGF-beta1 on the MDA-MB-231 cell line probably acts through a mechanism independent of the effect of TGF-beta1 on oestrogen concentrations since this cell line is hormone unresponsive.
In 2015, a remarkably high density bloom of Alexandrium minutum occurred in Sungai Geting, a semi-enclosed lagoon situated in the northeast of Peninsular Malaysia, causing severe discoloration and contaminated the benthic clams (Polymesoda). Plankton and water samples were collected to investigate the mechanisms of bloom development of this toxic species. Analysis of bloom samples using flow cytometry indicated that the bloom was initiated by the process of active excystment, as planomycetes (>4C cells) were observed in the early stage of the bloom. Increase in planozygotes (2C cells) was evident during the middle stage of the bloom, coinciding with an abrupt decrease in salinity and increase of temperature. The bloom was sustained through the combination of binary division of vegetative cells, division of planozygotes, and cyst germination through continuous excystment. Nutrient depletion followed by precipitation subsequently caused the bloom to terminate. This study provides the first continuous record of in situ life-cycle stages of a natural bloom population of A. minutum through a complete bloom cycle. The event has provided a fundamental understanding of the pelagic life-cycle stages of this tropical dinoflagellate, and demonstrated a unique bloom development characteristic shared among toxic Alexandrium species in coastal embayments.
Forty-eight patients with breast carcinoma were subjected to four quadrant fine needle aspiration (FNA) cytology examination of the ipsilateral and contralateral breast in an attempt to detect any accompanying benign proliferative lesion. Mastectomy of ipsilateral and open biopsy of contralateral breast provided material for histopathological study. Cytological evidence of epithelial proliferation was found in 8 (16.6%) cases which included atypical lobular hyperplasia (ALH), lobular neoplasia in-situ (LNIS), atypical ductal hyperplasia (ADH), and proliferative disease without atypia (PDWA). In lobular proliferative lesions, cytological smears showed configurations of cells that resembled filled up or expanded lobular units. The cytology was not distinctive enough to distinguish the sub-types of lobular proliferations. Likewise, the presence of ductal alterations could be suggested by cytological study but the distinction of proliferative disease without atypia (PDWA) from atypical ductal hyperplasia (ADH) was not possible on a cytological basis.
Oestrone sulphate is a major source of active oestrogens in the breast. It is converted to oestrone by oestrone sulphatase. Breast cyst fluid (BCF) is a rich source of sex hormones and growth factors. BCF obtained from British women has been shown to inhibit oestrone sulphatase activity in the MCF-7 oestrogen-receptor-positive breast cancer cell line. The aim of the present study was to assess whether BCF obtained from Malaysian women inhibited oestrone sulphatase activity in the MCF-7 and MDA-MB-231 breast cancer cell lines. The cell lines were grown in supplemented Dulbecco's Modified Eagle Medium for 3 days, following which a 3-day incubation with sterilised BCF was carried out. At the end of the treatment period the cell monolayers were assayed for oestrone sulphatase activity and the number of cell nuclei counted on a Coulter Counter. BCF was also fractionated on a Bio-Sil SEC 125-5 column by HPLC and the effects of the fractions collected on oestrone sulphatase activity in the MDA-MB-231 cell line were assessed. All 18 samples of BCF tested inhibited cell growth in the MDA-MB-231 cell line while 8 out of 10 samples inhibited MCF-7 cell growth; 15 out of 18 BCF samples inhibited oestrone sulphatase activity in the MDA-MB-231 cell line whereas 5 out of 10 samples stimulated oestrone sulphatase activity in the MCF-7 cell line. HPLC fractions corresponding to molecular weights of > 158 kDa and 28 kDa were found to inhibit oestrone sulphatase activity in the MDA-MB-231 cell line. Further work is required to fully characterise these substances as they may have roles to play in the prevention of breast cancer.
Breast cancer is the most common cancer in women worldwide. The growth of breast cancer cells is either hormone-dependent or hormone-independent. Both types are represented in vitro by the estrogen-receptor positive (ER+) MCF-7 and the estrogen-receptor negative (ER-) MDA-MB-231 cell lines, respectively. The pS2 gene is an estrogen-regulated gene and serves as a marker for the ER+ tumours. Carotenoids are pigments with anti-cancer properties besides having pro-vitamin A, antioxidant and free-radical quenching effects. This study was designed firstly, to compare the effect of palm oil carotene concentrate with retinoic acid on the growth of the ER+ MCF-7 and the ER- MDA-MB-231 cells; and secondly to evaluate the effect of the palm oil carotene concentrate on the regulation of pS2 mRNA. The growth experiments were performed with monolayer cells seeded in phenol red free RPMI 1640 culture media and subsequently treated with varying concentrations of either retinoic acid or palm oil carotenoids. The cell numbers were determined at the start of each experiment and then at successive time intervals. The results showed that the palm oil carotene concentrate caused dose-dependent inhibition of estradiol-stimulated growth of MCF-7 cells but did not affect the proliferation of MDA-MB-231 cells. Retinoic acid caused similar, albeit more potent effects, as significant inhibition was observed at lower concentrations than the palm oil carotenoids. In the pS2 gene expression experiment, cell monolayers were treated with the carotene concentrate (10(-6) M), either with or without supplemented estradiol (10(-8) M), and subsequently the RNA was extracted. Northern blotting was performed and the regulation of pS2 mRNA determined using a 32P-labelled pS2 cDNA probe. The results showed that the palm oil carotene concentrate did not affect the expression of pS2 mRNA and are therefore independent of the estrogen-regulated pathway.
Oestrogen is important in the development of breast cancer. Oestrogen receptor positive breast cancers are associated with a better prognosis than oestrogen-receptor negative breast cancers since they are more responsive to hormonal treatment. Oestrone sulphate acts as a huge reservoir for oestrogens in the breast. It is converted to the potent oestrogen, oestradiol (E(2)) by the enzymes oestrone sulphatase and oestradiol-17beta hydroxysteroid dehydrogenase (E(2)DH). Retinoic acid and carotenoids have been shown to have chemopreventive activity against some cancers. The aim of our study was to determine and compare the effects of retinoic acid and palm oil carotenoids on growth of and oestrone sulphatase and E(2)DH activities in the oestrogen receptor positive, MCF-7 and oestrogen receptor negative, MDA-MB-231 breast cancer cell lines. Retinoic acid and carotenoids inhibited MCF-7 cell growth but had no effect on MDA-MB-231 cell growth. Both retinoic acid and carotenoids stimulated oestrone sulphatase activity in the MCF-7 cell line. E(1) to E(2) conversion was inhibited by 10(-7) M carotenoids but was stimulated at 10(-6) M in the MCF-7 cell line. Retinoic acid had no effect on E(1) to E(2) conversion at 10(-7) M but stimulated E(1) to E(2) conversion at 10(-6) M. Retinoic acid and carotenoids had no effect on E(2) to E(1) conversion in the MCF-7 cell line. Retinoic acid stimulated E(1) to E(2) conversion in the MDA-MB-231 cell line but had no effect on oestrone sulphatase activity or E(2) to E(1) conversion in this cell line. Both oestrone sulphatase and E(2)DH activity were not affected by carotenoids in the MDA-MB-231 cell line. In conclusion, retinoic acid and carotenoids may prevent the development of hormone-dependent breast cancers since they inhibit the growth of the MCF-7 cell line.
Oestrogens play an important role in the development of breast cancer. A very important source of active oestrogens in the breast is oestrone sulphate which is converted to oestrone by oestrone sulphatase. The aim of this study was to assess the effects of IGF-I and IGF-II on oestrone sulphatase activity in, as well as cell growth of, MCF-7 and MDA-MB-231 human breast cancer cell lines. Cells were grown in supplemented DMEM and treated with varying concentrations of IGFs. At the end of the treatment period, intact cell monolayers were washed and assayed for oestrone sulphatase activity and the number of cell nuclei determined on a Coulter Counter. Oestrone sulphatase activity was significantly stimulated by IGF-I and II at concentrations of 100 ng/ml and 200 ng/ml in MCF-7 cells. IGF-I had no effect on oestrone sulphatase activity in MDA-MB-231 cells over the range of concentrations tested. Significant inhibition of oestrone sulphatase was observed in MDA-MB-231 cells at higher concentrations of IGF-II (50 ng/ml, 100 ng/ml and 200 ng/ml). Both IGF-I and IGF-II at higher concentrations (100 ng/ml and 200 ng/ml) significantly inhibited MCF-7 and stimulated MDA-MB-231 cell growth. Since IGF-I and II have effects on cell growth and oestrone sulphatase activity in breast cancer cell lines they may play a role in the development and progression of human breast cancer.
Oestrogens play an important role in the development of breast cancer. Oestrone sulphate (E1S) acts as a huge reservoir of oestrogens in the breast and is converted to oestrone (E1) by oestrone sulphatase (E1STS). E1 is then reversibly converted to the potent oestrogen, oestradiol (E2) by oestradiol-17beta hydroxysteroid dehydrogenase (E2DH). The aim of this study was to assess the effects of transforming growth factor-beta1 (TGFbeta1), insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) on cell growth, E1STS and E2DH activities in the MCF-7 and MDA-MB-231 human breast cancer cell lines. TGFbeta1, IGF-I and IGF-II alone or in combination inhibited cell growth of both cell lines but no additive or synergistic effects were observed. The treatments significantly stimulated E1STS activity in the MCF-7 cell line, except for TGFbeta1 alone and TGFbeta1 and IGF-I in combination, where no effects were seen. Only TGFbeta1 and IGF-II acted synergistically to stimulate E1STS activity in the MCF-7 cells. There was no significant effect on E1STS activity in the MDA-MB-231 cells with any of the treatments. In the MCF-7 cells, TGFbeta1 and IGF-I, IGF-I and IGF-II, and TGFbeta1, IGF-I and IGF-II acted synergistically to stimulate the reductive E2DH activity, while only TGFbeta1, IGF-I and IGF-II synergistically stimulated the oxidative E2DH activity. There were no additive or synergistic effects on both oxidative and reductive E2DH activities in the MDA-MB-231 cells. In conclusion, TGFbeta1, IGF-I and IGF-II may have effects on oestrogen metabolism, especially in the MCF-7 cell line where they stimulated the conversion of E1S to E1 and E1 to E2 and, thus, may have roles to play in the development of breast cancer.
Trichomonas vaginalis, a flagellated protozoan parasite, is commonly found in the genitourinary tract of humans. Its mode of reproduction has always been reported to be binary fission. The high parasite numbers seen in a relatively short period in in vitro cultures led us to believe that there must be other modes of reproduction. The present study for the first time provides transformational evidence at the ultrastructural level seen in tropohozoites of T. vaginalis undergoing a multiple asexual mode of reproduction. The findings show that the single cell with a nucleus is capable of dividing to as many as eight nuclei within the cytoplasmic body. Before the commencement of division, the nucleus remained round or ovoid in shape with condensed chromatin masses and only a few endoplasmic reticula surrounding the nucleus. During the division, the nucleus started to elongate and become irregular in shape with visible chromatin masses condensing with the accumulation of numerous endoplasmic reticula. Nuclear division gave rise to as many as eight nuclei within a cell, which could be seen to be connected by numerous endoplasmic reticula. In addition, a high number of hydrogenosomes and vacuoles can be seen in multinucleated T. vaginalis compared with single nucleated T. vaginalis. This study confirms that multiple modes of nuclear division do exist in T. vaginalis and are a precursor to progeny formation.
The karyotypes have been determined of 16 of the 32 species of the genus Varanus, including animals from Africa, Israel, Malaya and Australia. A constant chromosome number of 2n = 40 was observed. The karyotype is divided into eight pairs of large chromosomes and 12 paris of microchromosomes. A series of chromosomal rearrangements have become established in both size groups of the karyotype and are restricted to centromers shifts, probably caused by pericentric inversion. Species could be placed in one of six distinct karyotype groups which are differentiated by these rearrangements and whose grouping does not always correspond with the current taxonomy. An unusual sex chromosome system of the ZZ/ZW type was present in a number of the species examined. The evolutionary significance of these chromosomal rearrangements, their origin and their mode of establishment are discussed and related to the current taxonomic groupings. The most likely phylogenetic model based on chromosome morphology, fossil evidence and the current distribution of the genus Varanus is presented.