Malaria parasites, Plasmodium can infect a wide range of hosts including humans and rodents. There are two copies of mitogen activated protein kinases (MAPKs) in Plasmodium, namely MAPK1 and MAPK2. The MAPKs have been studied extensively in the human Plasmodium, P. falciparum. However, the MAPKs from other Plasmodium species have not been characterized and it is therefore the premise of presented study to characterize the MAPKs from other Plasmodium species-P. vivax, P knowlesi, P berghei, P chabaudi and P.yoelli using a series of publicly available bioinformatic tools. In silico data indicates that all Plasmodium MAPKs are nuclear-localized and contain both a nuclear localization signal (NLS) and a Leucine-rich nuclear export signal (NES). The activation motifs of TDY and TSH were found to be fully conserved in Plasmodium MAPK1 and MAPK2, respectively. The detailed manual inspection of a multiple sequence alignment (MSA) construct revealed a total of 17 amino acid stack patterns comprising of different amino acids present in MAPK1 and MAPK2 respectively, with respect to rodent and human Plasmodia. It is proposed that these amino acid stack patterns may be useful in explaining the disparity between rodent and human Plasmodium MAPKs.
Kajian ini melibatkan pemantauan perkembangan parasitemia dan taburan morfologi Plasmodium berghei sewaktu infeksi parasit dalam mencit, serta penentuan kesan infeksi P. berghei ke atas pengisyaratan MAP kinase eritrosit perumah. Analisis mikroskop ke atas slaid calitan darah terwarna-Giemsa yang disediakan daripada mencit terinfeksi-P. berghei (strain PZZ1/00) menunjukkan darjah parasitemia mencapai sehingga 70% dalam masa dua minggu selepas penyuntikan parasit. Morfologi cecincin dan trofozoit parasit dicerap dengan jelas sepanjang tempoh infeksi manakala morfologi skizon parasit hanya dicerap dengan ketara selepas hari ketiga selepas penyuntikan parasit. Pemblotan Western [antibodi primer: anti-MAP kinase (ERK-1/2 tak terfosfat) monoklon; antibodi sekunder: anti-IgG, poliklon terkonjugat-HRP] ke atas protein sitosol eritrosit terinfeksi-P. berghei (70% parasitemia) susulan pemisahan SDS-PAGE menunjukkan bahawa keamatan protein imunoreaktif-MAP kinase eritrosit berberat molekul 42 dan 44 kDa didapati meningkat secara signifikan (p<0.05) pada 70% iaitu peningkatan sebanyak 21.5% dan 22.3% masing-masing berbanding sampel kawalan tanpa infeksi. Samada kesan infeksi P. berghei (70% parasitemia) ke atas pengisyaratan MAP kinase perumah ini berkaitan dengan pengaktifan enzim ini perlu dikaji dengan lebih lanjut.
Penemuan sasaran dadah antikoksidia baharu merupakan antara usaha yang diperlukan untuk mengawal penyakit koksidiosis ayam yang disebabkan oleh spesies Eimeria. Dalam kajian ini, serpihan yang mengekodkan glikogen sintase kinase-3 (GSK-3) Eimeria tenella putatif telah diamplifikasi daripada cDNA E. tenella. Hasil pemadanan homologi menunjukkan jujukan GSK-3 E. tenella yang terjana mempunyai padanan yang tinggi dengan jujukan GSK-3 organisma lain. Domain terpulihara GSK-3 dan residu yang penting untuk aktiviti GSK-3 juga diramalkan hadir dalam jujukan GSK-3 E. tenella. Analisis struktur sekunder serta pemodelan homologi menunjukkan pembahagian struktur protein kepada domain bebenang beta pada hujung N dan domain heliks alfa pada hujung C, yang merupakan ciri enzim GSK-3. Kesemua hasil analisis ini menyokong bahawa jujukan yang dikaji mengekodkan protein GSK-3 dalam E. tenella. Walaupun darjah keterpuliharaan adalah tinggi, namun terdapat perbezaan yang bermakna diperhatikan antara GSK-3 E. tenella dan perumahnya. Residu Ser 9 yang dilaporkan penting untuk perencatan aktiviti GSK-3 didapati tidak terpulihara dalam GSK-3 E. tenella. Memandangkan Ser 9 merupakan tapak pemfosfatan bagi GSK-3β dalam haiwan vertebrata, ketiadaan residu ini dalam jujukan GSK-3 E. tenella mencadangkan bahawa pengawalaturan GSK-3 E. tenella melibatkan tapak pemfosfatan dan mekanisme yang berbeza. Tambahan pula, hasil analisis filogenetik menunjukkan bahawa GSK-3 E. tenella mempunyai pertalian yang rapat dengan protein GSK-3 tumbuh-tumbuhan. Analisis superposisi GSK-3 E. tenella dengan GSK-3β Homo sapiens pula menunjukkan bahawa perencat GSK-3 mampu berinteraksi dengan protein GSK-3 E. tenella. Keputusan kajian ini mencadangkan bahawa GSK-3 E. tenella mempunyai potensi untuk diperkembangkan sebagai sasaran dadah antikoksidia.
Gynura procumbens, locally known in Malaysia as Sambung Nyawa is a medicinal plant belonging to the Asteraceae (Compositae) family. G. procumbens have been traditionally used by the local and indigenous populations to treat an array of ailments ranging from skin conditions and fever to kidney disease, inflammation and diabetes. In the present investigation, aqueous and ethanol extracts of G. procumbens were evaluated for anti-plasmodial activities in vitro and in vivo. Survival of two chloroquine-sensitive strains of malarial parasites; rodent Plasmodium berghei NK65 and human Plasmodium falciparum 3D7 was determined following incubations in vitro with extracts. Based on parasite lactate dehydrogenase (pLDH) assay, both extracts were shown to inhibit parasite proliferation to varying degrees. The aqueous extract was more potent than the ethanol extract at suppressing growth of both parasites in vitro; each displaying IC50 values of 12.40 ± 6.02 and 14.38 ± 7.53 μg/mL towards P. berghei NK65; and 25.69 ± 4.34 and 42.23 ± 7.19 μg/mL towards P. falciparum 3D7, respectively. The aqueous extract was found to be selective for P. falciparum (Selectivity Index 64.30). Four-day suppressive tests in ICR mice showed dose-dependent chemo-suppressive activities of both plant extracts tested towards P. berghei NK65. Daily intra-peritoneal injections of the aqueous extract of G. procumbens at 25, 50 or 100 mg/kg for four consecutive days showed chemo-suppression of 50.42 ± 3.17, 65.95 ± 5.48 and 81.92 ± 3.07%, respectively. At the same dosages, the ethanol plant extract resulted in 44.97 ± 3.44, 55.21 ± 3.87 and 64.44 ± 4.05% chemo-suppression respectively. At 250 mg/kg/day, only the aqueous plant extract gave >90% chemo-suppression (93.06 ± 5.46%). Treatment of P. berghei-infected mice with extracts improved the median survival time compared to non-treated infected mice. This represents the first report showing anti-plasmodial activity of G. procumbens.
Burkholderia pseudomallei, the causative agent of melioidosis, is an intracellular pathogen capable of invading and multiplying in both phagocytic and non-phagocytic cells. Infection results in an inflammatory response involving production of both pro- and anti-inflammatory cytokines. The cellular mechanism regulating this response, believed to play an important role in the pathogenesis of meliodoisis, is not fully understood. In recent years, glycogen synthase kinase-3 (GSK3) has been shown to assume a pivotal role in regulating production of these cytokines. Bacterial infection of host cells activates Toll-like receptors (TLRs) and results in the phosphorylation of GSK3β through activation of the phosphoinositide 3-kinase (PI3K) pathway. In this study, we investigated the effects of GSK3 inhibition in regulating B. pseudomallei-induced inflammatory response in macrophages and A549 epithelial lung cells. Our results showed that infection of cells with B. pseudomallei resulted in the increase of anti-inflammatory cytokine, IL-10 and pro-inflammatory cytokine, TNF-α. Pre-treatment of infected cells with GSK3 inhibitors caused further increase in the level of IL-10 but a significant decrease in TNF-α. These changes corresponded with the detection of phosphorylated GSK3β in infected cells treated with LiCl; suggesting that modulation of inflammatory response in B. pseudomallei infection involves phosphorylation of GSK3β (Ser 9). This could explain our observations from the invasion assays that pre-treatment of B. pseudomallei-infected cells with GSK3 inhibitors resulted in decreased intracellular replication of bacteria within macrophages and A549 epithelial lung cells. In summary, our results demonstrate a regulatory function of GSK3 in the modulation of cytokine levels during B. pseudomallei infection.
Hepatic phosphoprotein levels are altered in mouse liver as a manifestation of bacteria, virus or parasite infection. Identification of signaling pathways mediated by these hepatic proteins contribute to the current understanding of the mechanism of pathogenesis in malarial infection. The present study was undertaken to evaluate the changes in hepatic phosphoprotein levels during Plasmodium berghei infection. Our study revealed changes in levels of three hepatic phosphoproteins following P. berghei infection compared to non-infected controls. Peptide fragment sequence analysis using tandem mass spectrometry (MS/MS) showed these hepatic proteins to be homologs to haemoglobin beta (HBB), class
Pi glutathione S-tranferase (GSTPi) and carbonic anhydrase III (CAIII) proteins of Mus musculus species respectively from the NCBInr sequence database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis predicted the involvement of these proteins in specific pathways in Mus musculus species; GSTPi in glutathione and drug metabolism and CAIII in nitrogen metabolism. This shows that P. berghei infection affects similar signaling pathways as those reported in other pathogenic infections such as that related to GSTPi and CAIII in response to oxidative stress.
Gynura procumbens is a medicinal plant, traditionally used to treat inflammation and fever. A yeast-based assay detected GSK3β-inhibitory activity in the aqueous extract of G. procumbens. GSK3β is now known to have a central role in the modulation of host inflammatory response during bacterial infections. In this study, we investigated the involvement of GSK3β in the anti-malarial and anti-inflammatory effects of an aqueous extract of G. procumbens. Our results showed that G. procumbens inhibited growth of P. falciparum 3D7. Consecutive four-day administration of 250 mg/kg body weight (b.w.) G. procumbens resulted in strong chemosuppression and improved survivability in P. berghei-infected mice. B. pseudomallei-infected mice treated with G. procumbens (50 mg/kg b.w.) showed increased survivability. TNF-α and IFN-γ levels in liver and serum of B. pseudomallei-infected mice were lowered by G. procumbens treatment. IL-10 level was higher in serum of G. procumbens-administered infected mice. G. procumbens treatment of P. berghei- and B. pseudomallei-infected animals each resulted in increased hepatic GSK3β (Ser9) phosphorylation. It is noteworthy that kaempferol (one of the compounds in G. procumbens) also inhibited the growth of P. falciparum 3D7; showed strong chemosuppression and improved survivability in P. berghei-infected mice at 5 mg/kg b.w. B. pseudomallei-infected mice treated with kaempferol (10 mg/kg b.w.) showed improved survivability. Concomitantly, the described effects due to kaempferol also involved enhanced GSK3β (Ser9) phosphorylation as observed with G. procumbens. In summary, the observed anti-malarial and anti-inflammatory effects of G. procumbens involved inhibition of GSK3β and kaempferol may in part be responsible for the pharmacological effects.
Gynura procumbens, a medicinal plant locally known as Sambung nyawa, is widely used traditionally in South East Asia as a remedy to alleviate symptoms of various ailments including diabetes mellitus. Previous investigators reported that the ethanol plant extract was hypoglycemic in streptozotocin (STZ)-induced diabetic rats and suggested the blood glucose-reducing effect could be due to G. procumbens mimicking insulin effects. Our present study aimed to reevaluate the hypoglycemic effect(s) of G. procumbens and to ascertain the involvement of glycogen synthase kinase (GSK3), a key component of insulin biosignaling in the plant activity. A cell-based assay showed that the three G. procumbens fractions tested did not exhibit anti-GSK3 activities. Oral administrations of hexane, ethyl acetate, n-butanol fractions of G. procumbens (250 mg/kg b.w.) and Glibenclamide (5 mg/kg b.w.) for 14 days in STZ-induced diabetic rats reduced blood glucose levels by 29.7%, 60.1%, 33.5% and 61.7% (p<0.05), respectively compared to diabetic control. Liver glycogen contents were elevated (p<0.05), following administration of the three fractions of
G. procumbens in diabetic rats when analysed after 14 days of treatment. Western blot analysis also showed GSK3β in the liver of G. procumbens fractions-treated and Glibenclamide-treated animals were phosphorylated at Ser-9. The above findings indicated that the hypoglycemic action of G. procumbens fractions resulted in the inactivation of
GSK3β in liver of diabetic rats. Since G. procumbens fractions did not exhibit GSK3 inhibitory properties, it is possible that the hypoglycemic action of the fractions observed here could be due to direct or indirect effects on upstream components of the insulin biosignaling pathway.