Fibropreventive and Antifibrotic Effects of Uncaria gambir on Rats with Pulmonary Fibrosis

Pulmonary fibrosis causes scar tissue formation that disrupts the functioning of the lungs. Uncaria gambir (Hunter) Roxb (hereafter gambir)—a plant native to West Sumatra in Indonesia—contains flavonoid (+)-catechin and has strong antioxidant activity, and it can be used to combat pulmonary fibrosis. This random in vivo experimental study analyzed the antifibrotic effect of gambir on the lungs of rats with bleomycin-induced fibrosis. The subjects were 10 groups of 10-week-old male rats weighing around 200–250 g. All groups were terminated at the end of the seventh week or on day 50. The lungs were cleaned, and tissues were taken to analyze inflammatory cell counts and TGF-β1 levels using bronchoalveolar lavage (BAL) with ELISA; type I collagen and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels using immunohistochemistry (IHC); and activation of NF-κB using ELISA and Western blot assays. The most severe histopathological characteristic based on the modified Ashcroft score was in the bleomycin group (BG), whereas the mildest was in the 262 mg/kg of bodyweight antifibrotic gambir-dosed group (AF G262). The results showed a significant difference in the BAL inflammatory cell count (p = 0.017; p < 0.05). AF G262 differed most from the other antifibrotic groups in terms of the number of inflammatory cells (0.63), TGF-β1 levels (3.80), and NF-κB levels (0.48), followed by the 131 mg/kg of bodyweight antifibrotic gambir-dosed group (AF G131), which also differed most from other antifibrotic groups in terms of NF-κB (0.48), TIMP-1 (11.74), and collagen I (14.50) levels. Western blot analysis showed that the fibropreventive and antifibrotic groups had a specific band size of p65, whereas no specific band binding existed in the control group. This study concluded that the administration of AF G262 could improve fibrosis by lysing the extracellular matrix (ECM) in rat lungs.

Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method

Paramecium spp. is types of free-living protists that live in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium’s unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

Rsp activates expression of the Cnt system in Staphylococcus aureus

Background The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Detection of Powdery Mildew Growth in Hazelnut Plant Using PCR

Powdery mildew is a serious disease of economically important hazelnut crop in Turkey. Hazelnut production has been extremely affected by the disease in terms of quality and quantity. The disease is caused by two different fungi, namely Erysiphe corylacearum and Phyllactinia guttata. E. corylacearum has been shown to be the responsible one predominantly for the recent economic damage. The fungi produce a mycelium network on hazelnut plants before they sporulate and visually detected. Early detection of these pathogens is important for management as well as understanding their spread and epidemics. In this study, a PCR assay was developed for the detection of both pathogens from hazelnut plant leaves by targeting their ribosomal DNA genes in their internal transcribed spacer (ITS) regions. Two sets of specific primers were designed for the detection of E. corylacearum and P. guttata at an early stage of infection. As a result of PCR, a specific band of 578 bp was observed. The amplicon sequencing confirmed the presence of only E. corylacearum, but not P. guttata. Therefore, this PCR-based test can identify plants that are infected with powdery mildew before they show any visual signs. From there, the infected plants can be treated or removed before the fungus has a chance to produce spores that infect neighboring plants. These results would help tackle the eradication of powdery mildew.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A twofold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

VARIASI GENETIK BEBERAPA SPESIES KAPAS (Gossypium sp.) BERDASARKAN KERAGAMAN POLA PITA ISOZIM

<p>ABSTRAK</p><p>Deskripsi aksesi-aksesi kapas berdasarkan karakter morfologinyatelah disusun berdasarkan descriptor list yang disusun oleh IBPGR, akantetapi marka genetik dari aksesi-aksesi tersebut belum diketahui. Penelitianini bertujuan untuk mempelajari keragaman pola-pita isozim Peroksidase(PER), Esterase (EST), dan Aspartate amino transferase (AAT) pada 19aksesi kapas dan kemiripan ke-19 aksesi kapas berdasarkan ketiga isozimtersebut. Penelitian ini dilaksanakan mulai bulan Februari 2008 di RumahKaca Fakultas Pertanian, Universitas Sebelas Maret Surakarta dan analisisisozim dilakukan di Laboratorium Biologi Tumbuhan, PAU Ilmu HayatIPB. Metode analisis yang digunakan adalah elektroforesis gel pati tipehorisontal dengan tiga sistem enzim, yaitu enzim peroksidase (PER),esterase (EST), dan aspartate amino transferase (AAT). Penelitianmenghasilkan data berupa pola pita isozim yang selanjutnya dibuat dalamdata biner. Data biner yang dihasilkan dibuat dalam persamaan matrik dandilanjutkan analisis gerombol dengan metode ‘UPGMA’ (Unweighted PairGroup Method Arithmetic Average) menggunakan fungsi SHAN padaProgram NTSYSpc versi 2.02. Hasil penelitian menunjukkan bahwaIsozim esterase dapat dijadikan marka genetik bagi Kanesia 1 (terbentuksatu pita spesifik) dan Kanesia 6 (satu pita spesifik); isozim peroksidasedapat dijadikan marka genetik bagi Kanesia 3 (dua pita pada kutub positif),aksesi-aksesi G. barbadense (dalam hal ini CTX-3 dan Giza-90 dua pitapada kutub positif) dan G. arboreum (empat pita pada kutub positif dansatu pita pada kutub negatif). Sedangkan isozim aspartat amino tranferasedapat dijadikan marka genetik bagi spesies G. herbaceum (dua pitaspesifik). Selain itu, terdapat kemiripan genetik antar aksesi kapasberdasarkan ketiga isozim (EST, PER, dan AAT). Pengelompokanberdasarkan ketiga isozim dari ke-19 aksesi kapas diketahui bahwa padajarak kemiripan 0,59 atau kemiripan 59% semua aksesi kapas menyatu,yang terbagi menjadi 2 kelompok. Kelompok pertama hanya terdiri aksesiKanesia 1 saja. Sedangkan Kelompok kedua terdiri atas aksesi-aksesiKanesia 2, Kanesia 3, Kanesia 6, Kanesia 4, Kanesia 10, Kanesia 7,Kanesia 11, Kanesia 12, M-5, Kanesia 8, Kanesia 9, Kanesia 15, AKA-5,Kanesia 13, Kanesia 14, CTX-3, Giza-90 dan Kanesia 5.</p><p>Kata kunci: Gossypium sp., keragaman genetik, pola pita isozim</p><p>ABSTRACT</p><p>Genetic Diversity of Cotton Species (Gossypium sp.)Based on Variation of Isozyme Banding Pattern</p><p>Morphological characters of cotton accessions have been describedbased on the descriptor list produced by IBPGR, but the genetic markersfor those accessions have not yet been known. This research aimed atstudying the diversity and similarity among 19 cotton accessions based onisozyme banding patterns of peroxidase (PER), esterase (EST), andaspartate amino transferase (AAT). Research was carried out in February2008 at the green house of Faculty of Agriculture, Sebelas MaretUniversity, Surakarta and the isozyme was analyzed in Plant BiologicalLaboratory, Biological Science PAU IPB. Samples were electrophoresedon horizontal type of potato extract gel and stained with three enzymesystems, i.e. peroxidase (PER), esterase (EST), and aspartate aminotransferase ( AAT). The isozyme bandings were scored and translated intobinary data, which was then used to deduce the similarity amongaccessions and to draw dendrogram by using 'UPGMA' (Unweighted PairGroup Method Arithmetic Average) method from the NTSYSPC softwareversion 2.02. Experimental results showed that isozyme esterase can beused as genetic marker for Kanesia 1 (one specific band) and Kanesia 6(one specific band). Isozyme peroxidase can be used as genetic marker forKanesia 3 (two bands at positive end), accessions G. barbadense i.e. CTX-3 and Giza-90 (two bands at positive end) and G. arboreum (four bands atpositive end and one band at negative). Isozyme aspartate aminotransferase can be used as genetic marker for spesies G. herbaceum (twospecific bands). Moreover, the similarity analysis among 19 cottonaccessions based on the three isozymes showed that at the similarity levelof 59%, all accessions are divided in two groups. The first group consistedof Kanesia 1 only. Whereas the second group consisted of accessionsKanesia 2, Kanesia 3, Kanesia 6, Kanesia 4, Kanesia 10, Kanesia 7,Kanesia 11, Kanesia 12, M-5, Kanesia 8, Kanesia 9, Kanesia 15, AKA-5,Kanesia 13, Kanesia 14, CTX-3, Giza-90, and Kanesia 5.</p><p>Key words: Gossypium sp., genetic diversity, isozyme banding pattern</p>