30 research outputs found
Cellular response to PorA-Loop4-derived peptides of Neisseria meningitidis
Get PDFNeisseria meningitidis (the meningococcus) is a major meningitis-causing bacterium and is known for its ability to breach the blood-brain barrier (BBB). Meningococci bind to the Laminin receptor (LAMR) on the surface of the vascular endothelium, which is part of the BBB. In a previous study, the meningococcal surface proteins PorA and PilQ were identified as the bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). PorA preferentially targets the 37 kDa laminin receptor precursor (37LRP) rather than the 67 kDa laminin receptor form (67LR) on the cell surface. It was confirmed that the PorA:37LRP interaction is mediated by PorA-Loop4 as deletion of this loop abrogates recruitment of 37LRP under meningococcal colonies. Using a cyclised peptide corresponding to PorA-Loop4 from N. meningitidis MC58, the PorA-Loop4:37LRP interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1-phase cell cycle arrest. The interaction of PorA-Loop4:37LRP was cytostatic, not cytotoxic. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells at S-phase and a corresponding increase in the G1 population.
Based on these results, it is hypothesised that PI3K/Akt pathway was affected by the interaction resulting in G1 arrest. The current study aimed to investigate the perturbations of key proteins’ expression and activation involved in the PI3K/Akt pathway (including Akt, GSK-3β, Cyclin D1, and CDK4) after the treatment of PorA-Loop4 peptide in HBMECs.
The work indicated that PorA-Loop4 caused perturbations of Cyclin D1/CDK4 as cell cycle regulators at G1 phase. Transcriptome analysis using qRT-PCR showed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunoblotting confirmed these results as the 24 h-treatment of PorA-Loop4 peptide caused the downregulation of Cyclin D1 and the upregulation of pCDK4 (Thr172).
PorA-Loop4 caused a blockade of PI3K/Akt signalling. Using ZSTK474 as a PI3K inhibitor, we observed that the effect of PorA-Loop4 on Cyclin D1 and pCDK4 (Thr172) was PI3K-dependent at both mRNA and protein levels.
Exposure of HBMECs to PorA-Loop4 peptide caused a downregulation of pAkt (Ser473) and an upregulation of pGSK-3β (Ser9) at the protein level 24 h post treatment. The upregulation of pGSK-3β (Ser9) was dependent to Akt, and the downregulation of Cyclin D1 was independent of GSK-3β.
Using the whole bacterial cells of N. meningitidis wild-type MC58 and its mutants lacking of PorA-Loop4, PorA, and/or PilQ, the role of PorA-Loop4, PorA and PilQ in influencing the PI3K/Akt pathway were investigated. Similar perturbations of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) occurred in PorA-Loop4 peptide treated cells and wild-type infected cells while the abrogation of the PorA-Loop4 erased the perturbations in HBMECs. Both PorA and PilQ have significant roles in the perturbation of pAkt (Ser473), pGSK-3β (Ser9), and Cyclin D1. However, the upregulation of pCDK4 (Thr172) was mediated by Loop4, not by other parts of the PorA, nor by PilQ.
Confocal microscopy showed the localisation of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the uninfected, wild-type-infected, and PorA-Loop4 mutant-infected HBMECs. The pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1 were localised mainly in the cytoplasm, while the pCDK4 (Thr172) was localised in cytoplasmic and nuclear regions. Confocal microscopy confirmed the immunoblotting results, as the diffuse pattern of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the wild-type infected cells were in contrast with the uninfected and the PorA-Loop4 mutant infected cells.
In summary, PorA-Loop4 causes the perturbation of PI3K/Akt pathway via Akt/GSK-3β/Cyclin D1/CDK4. The data presented in this thesis has extended knowledge of meningococcal-host pathogen interaction. A thorough understanding of PorA-Loop4 interaction with LAMR and its effect in the PI3K/Akt pathway may provide another strategy for the development of interventions against the meningococcus and perhaps other Gram-negative pathogens
Cellular response to PorA-Loop4-derived peptides of Neisseria meningitidis
Get PDFNeisseria meningitidis (the meningococcus) is a major meningitis-causing bacterium and is known for its ability to breach the blood-brain barrier (BBB). Meningococci bind to the Laminin receptor (LAMR) on the surface of the vascular endothelium, which is part of the BBB. In a previous study, the meningococcal surface proteins PorA and PilQ were identified as the bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). PorA preferentially targets the 37 kDa laminin receptor precursor (37LRP) rather than the 67 kDa laminin receptor form (67LR) on the cell surface. It was confirmed that the PorA:37LRP interaction is mediated by PorA-Loop4 as deletion of this loop abrogates recruitment of 37LRP under meningococcal colonies. Using a cyclised peptide corresponding to PorA-Loop4 from N. meningitidis MC58, the PorA-Loop4:37LRP interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1-phase cell cycle arrest. The interaction of PorA-Loop4:37LRP was cytostatic, not cytotoxic. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells at S-phase and a corresponding increase in the G1 population.
Based on these results, it is hypothesised that PI3K/Akt pathway was affected by the interaction resulting in G1 arrest. The current study aimed to investigate the perturbations of key proteins’ expression and activation involved in the PI3K/Akt pathway (including Akt, GSK-3β, Cyclin D1, and CDK4) after the treatment of PorA-Loop4 peptide in HBMECs.
The work indicated that PorA-Loop4 caused perturbations of Cyclin D1/CDK4 as cell cycle regulators at G1 phase. Transcriptome analysis using qRT-PCR showed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunoblotting confirmed these results as the 24 h-treatment of PorA-Loop4 peptide caused the downregulation of Cyclin D1 and the upregulation of pCDK4 (Thr172).
PorA-Loop4 caused a blockade of PI3K/Akt signalling. Using ZSTK474 as a PI3K inhibitor, we observed that the effect of PorA-Loop4 on Cyclin D1 and pCDK4 (Thr172) was PI3K-dependent at both mRNA and protein levels.
Exposure of HBMECs to PorA-Loop4 peptide caused a downregulation of pAkt (Ser473) and an upregulation of pGSK-3β (Ser9) at the protein level 24 h post treatment. The upregulation of pGSK-3β (Ser9) was dependent to Akt, and the downregulation of Cyclin D1 was independent of GSK-3β.
Using the whole bacterial cells of N. meningitidis wild-type MC58 and its mutants lacking of PorA-Loop4, PorA, and/or PilQ, the role of PorA-Loop4, PorA and PilQ in influencing the PI3K/Akt pathway were investigated. Similar perturbations of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) occurred in PorA-Loop4 peptide treated cells and wild-type infected cells while the abrogation of the PorA-Loop4 erased the perturbations in HBMECs. Both PorA and PilQ have significant roles in the perturbation of pAkt (Ser473), pGSK-3β (Ser9), and Cyclin D1. However, the upregulation of pCDK4 (Thr172) was mediated by Loop4, not by other parts of the PorA, nor by PilQ.
Confocal microscopy showed the localisation of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the uninfected, wild-type-infected, and PorA-Loop4 mutant-infected HBMECs. The pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1 were localised mainly in the cytoplasm, while the pCDK4 (Thr172) was localised in cytoplasmic and nuclear regions. Confocal microscopy confirmed the immunoblotting results, as the diffuse pattern of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the wild-type infected cells were in contrast with the uninfected and the PorA-Loop4 mutant infected cells.
In summary, PorA-Loop4 causes the perturbation of PI3K/Akt pathway via Akt/GSK-3β/Cyclin D1/CDK4. The data presented in this thesis has extended knowledge of meningococcal-host pathogen interaction. A thorough understanding of PorA-Loop4 interaction with LAMR and its effect in the PI3K/Akt pathway may provide another strategy for the development of interventions against the meningococcus and perhaps other Gram-negative pathogens
sodA and gap Genes as Markers for The Identification of Staphylococcus capitis
Get PDFBACKGROUND: Rapid and accurate identification of Staphylococcus capitis is required to provide a better prognosis for endocarditis patients and tackle the emergence of multidrug resistant strains of the bacteria in hospitals. The current study was aimed to develop polymerase chain reaction (PCR) assay for specific identification of S. capitis using sodA and gap genes as markers.METHODS: Five sequences of sodA and sixteen sequences of gap registered in GeneBank were analysed using bioinformatic tools. PCR primers were designed based on the conserved and specific regions of sodA and gap. Four clinical isolates of S. capitis (named no. 56-59) and six reference strains of coagulase-negative staphylococci (CoNS) species including S. epidermidis ATCC 35984, S. epidermidis 48951I/09, S. lugdunensis 44987/09, S. sciuri 109645I/08, S. warneri 135612/09, S. hominis 114202/08 were used to validate the conventional PCR system.RESULTS: The current PCR system only amplified the DNA template of S. capitis. Current primers specifically targeted S. capitis as the agarose images only showed bands from S. capitis samples.CONCLUSION: The sodA and gap genes might serve as effective markers for identification of S. capitis using conventional PCR. The PCR assay in the current study was able to identify five clinical isolates of S. capitis accurately without mispriming, misamplification and misidentification. The PCR system was also able to discriminate other CoNS including S. epidermidis, S. lugdunensis, S. sciuri, S. warneri and S. hominis.KEYWORDS: Staphylococcus, S. capitis, sodA, gap, PC
Isolation and identification of substances with anti-hepatitis c virus activities from kalanchoe pinnata
No full textPorA-Loop4 derived peptides of Neisseria meningitidis cause a G1 cell cycle arrest through the Akt signalling pathway in human brain microvascular endothelial cells
No full textIN VITRO AND IN VIVO STUDIES OF ANTIHYPERURICEMIC AND ANTIOXIDANT ACTIVITY FROM BULBS OF BAWANG TIWAI (ELEUTHERINE PALMIFOLIA (L.) MERR.) FROM INDONESIA
No full textObjectives: This study would like to investigate the in vitro antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl assay and in vitro xanthine oxidase activity of the bulbs. This study performs in vivo assays to study the antihyperuricemic activity and antioxidant in the hyperuricemic rat through plasma malondialdehyde measurement. Method: The study was conducted by testing the fresh bulbs of bawang tiwai (Eleutherine palmifolia (L.) Merr. with chemical solvent of ethanol 70% to extract the bulbs. Allopurinol and Vitamin C were used as positive control for the antihyperuricemic assay and antioxidant assay, respectively. Other chemical substances were also used in this study. This study used chicken extract (Brands) 20 ml/kg/body weight to induce the level of uric acid in the blood serum, and potassium oxonate (Sigma 156124) to inhibit the uricase in rats. Results: The results show that the levels of uric acid were measured using spectrophotometer with dichloro-hydroxybenzen sulfonate (Biolabo) a as reagent. The ethanol extract of bawang tiwai (EBT) (E. palmifolia (L.) Merr) was potential to reduce uric acid level at 140, 280, and 560 mg/kg body weight, but possibly without inhibition against xanthine oxydase activity. Conclusion: All doses of EBT could inhibit lipid peroxidation in hyperuricemic condition caused by high purine diet in 14 days
Isolation and identification of substances with anti-hepatitis c virus activities from kalanchoe pinnata
No full textDon't Drink Your Milk : Mengungkap Fakta Mengejutkan di Balik Mitos Kehebatan Susu
No full textKita harus minum segelas susu sehari.Susu memenuhi semua kebutuhan gizi anak.Susu tinggi kalsium dapat mencegah osteoporosis.Slogan-slogan seperti itu tak asing lagi di telinga kita. Saking hebatnya, susu sapi sering dijadikan solusi berbagai masalah kesehatan. Beraneka ragam susu pun muncul, misalnya susu khusus untuk bayi, untuk anak yang sulit makan, untuk remaja yang ingin tumbuh tinggi, hingga untuk lansia yang ingin mencegah pengeroposan tulang.Faktanya, susu dibutuhkan tubuh hanya dalam jumlah sedikit. Alih-alih bermanfaat, susu justru bisa menyebabkan anemia, diare, bronkhitis, leukemia, multiple sclerosis, kerusakan gigi, ruam kulit, serta beberapa penyakit berbahaya lainnya.Buku ini memaparkan berbagai fakta tentang susu yang perlu Anda ketahui serta solusi alternatifnya. Jika Anda dan keluarga masih minum susu, Anda wajib membaca buku ini.xiv+180hlm.;17x19c
IN VITRO AND IN VIVO STUDIES OF ANTIHYPERURICEMIC AND ANTIOXIDANT ACTIVITY FROM BULBS OF BAWANG TIWAI (ELEUTHERINE PALMIFOLIA (L.) MERR.) FROM INDONESIA
Get PDFObjectives: This study would like to investigate the in vitro antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl assay and in vitro xanthine oxidase activity of the bulbs. This study performs in vivo assays to study the antihyperuricemic activity and antioxidant in the hyperuricemic rat through plasma malondialdehyde measurement. Method: The study was conducted by testing the fresh bulbs of bawang tiwai (Eleutherine palmifolia (L.) Merr. with chemical solvent of ethanol 70% to extract the bulbs. Allopurinol and Vitamin C were used as positive control for the antihyperuricemic assay and antioxidant assay, respectively. Other chemical substances were also used in this study. This study used chicken extract (Brands) 20 ml/kg/body weight to induce the level of uric acid in the blood serum, and potassium oxonate (Sigma 156124) to inhibit the uricase in rats. Results: The results show that the levels of uric acid were measured using spectrophotometer with dichloro-hydroxybenzen sulfonate (Biolabo) a as reagent. The ethanol extract of bawang tiwai (EBT) (E. palmifolia (L.) Merr) was potential to reduce uric acid level at 140, 280, and 560 mg/kg body weight, but possibly without inhibition against xanthine oxydase activity. Conclusion: All doses of EBT could inhibit lipid peroxidation in hyperuricemic condition caused by high purine diet in 14 days
Financial Performance Assessment of PT Pelabuhan Indonesia II (Persero) in Comparison with other Port Companies Locally and Globally
Get PDFTwo-thirds of Indonesia is water. Thousands lined the island of Sabang to Merauke. As an archipelago, marine transportation plays an important role in the development of Indonesia. Supported by Indonesia's strategic location as it is situated at the crossroads of world trade routes. Therefore it required sea transportation system that is reliable and efficient. This can be achieved if the management of the navy, both shipping companies and port operator operates properly and professionally.This study focuses on the performance of the port operator, PT Pelabuhan Indonesia II, which has its head office in Jakarta. PT Indonesia II port operational areas of the company covers 10 provinces and it has three subsidiaries, affiliate one, two business units and one joint operation.The performance that discussed in this study focused on financial performance, since only a company with good financial performance that can perform the operations well too.This final will discuss the performance of PT Pe4labuhan Indonesia II and will also be compared with other companies in the same industry both locally and globally. The author will use some framework. In the end, come to the conclusion and recommendation for PT Pelabuhan Indonesia II to achieve better performance.As a conclusion, PT Pelabuhan Indonesia II almost rivaling global competition a leader in several assessment framework, with an optimal capital structure when the WACC minimized at 53% of debt and 47% of equity while the maximum of firm value of IDR 8,620,465,240,669.Keywords: Archipelago, Port, Performance, Comparison, Financial statements analysis
