Modulatory effect of the fruit rind extract of Garcinia indica Choisy against gamma radiation induced damage in human peripheral blood lymphocytes: a preliminary study.

PURPOSE: Nowadays people are exposed to radiation due to various reasons, including natural, diagnostic, occupational or accidental exposure. High level of exposure to ionizing radiation can be fatal to human body. Synthetic drugs used to prevent radiation-induced damage are toxic in nature. Recently, Herbal drugs are being screened as an alternative due to their mechanism of action. Garcinia indica (G. indica) is one of the traditional medicinal plant which contains phytochemicals having several medicinal properties. MATERIALS AND METHODS: In this study, G. indica extract was observed for its modulatory effect against 3 Gray (Gy) gamma radiation-induced damages in human peripheral blood lymphocytes. Various concentrations of G. indica extract ranging from 1 to 25 µg/mL was added to the blood post irradiation at 0 hr. Chromosomal aberration (CA) and Cytochalasin B blocked Micronuclei Cytome (CBMN) Assay were performed as per standard procedure. RESULTS: Radiomodulatory effect of Garcinia indica fruit rind extract (GIFRE) on CA and MN formation was observed in this study. Treatment of GIFRE did not affect the mitotic index. Positive inhibition percentages for dicentrics, total chromosomal aberrations and micronuclei were observed except for one instance. CONCLUSION: Owing to the various properties of Garcinia extracts, it makes it a potential candidate to be tested for its radiomodulatory effect. Based on the results observed in this preliminary study, it could act as a radiomodulatory agent. Radiomodulatory effect of GIFRE could possibly serve it as a potential herbal medicinal alternative to current drugs. However, results of this study need to be validated on larger sample size.

Chemotaxis of Burkholderia sp. strain SJ98 towards chloronitroaromatic compounds that it can metabolise.

BACKGROUND: Burkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2-chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds. RESULTS: Strain SJ98 could mineralize 2C4NP, 4C2NB and 5C2NB, and co-metabolically transform 2C3NP and 2C4NB in the presence of an alternative carbon source, but was unable to transform 4C2NP under these conditions. Positive chemotaxis was only observed towards the five metabolically transformed CNACs. Moreover, the chemotaxis was induced by growth in the presence of the metabolisable CNAC. It was also competitively inhibited by the presence of nitroaromatic compounds (NACs) that it could metabolise but not by succinate or aspartate. CONCLUSIONS: Burkholderia sp. strain SJ98 exhibits metabolic transformation of, and inducible chemotaxis towards CNACs. Its chemotactic responses towards these compounds are related to its previously demonstrated chemotaxis towards NACs that it can metabolise, but it is independently inducible from its chemotaxis towards succinate or aspartate.

Growth and physiological response of Arthrobacter protophormiae RKJ100 toward higher concentrations of o-nitrobenzoate and p-hydroxybenzoate.

Bioremediation of sites that are heavily contaminated with pollutant chemicals is a challenge as most of the microorganisms cannot tolerate higher concentrations of toxic compounds. Only a few strains of the genus Pseudomonas have been studied for their tolerance toward the higher concentrations of aromatic pollutant compounds, a phenomenon that is accompanied by various physiological changes. In the present study we have characterized the growth response and physiological changes (adaptations) of a Gram-positive bacterium, Arthrobacter protophormiae RKJ100, toward the higher concentrations of two aromatic compounds, viz. o-nitrobenzoate (ONB) and p-hydroxybenzoate (PHB). Arthrobacter protophormiae RKJ100 could utilize 30 mM ONB and 50 mM PHB as sole sources of carbon and energy. It was capable of growth on higher concentrations of ONB (up to 200 mM) and PHB (up to 150 mM) when the cells were pre-exposed to lower concentrations of these compounds. The adaptive responses shown by the organism during growth on higher concentrations of these compounds were evident from significant changes in cellular fatty acid profiles. In addition, Bacterial Adhesion To Hydrocarbon (BATH) assay and scanning electron microscopy showed substantial increase in cell surface hydrophobicity and decrease in cell size of A. protophormiae RKJ100 when grown on ONB and PHB as compared to succinate-grown cells.

Differential radiation effects in smokers--culture time dependence of the yield of gamma ray-induced chromosome damage in first division metaphases.

PURPOSE: Telomeric associations (TA) and unstable chromosomal aberration (CA) transmission through M1-M4 metphases (first to fourth division) in gamma-ray irradiated G0 lymphocytes in 2 smokers were examined, since TA in conventionally stained chromosomes were reported earlier as a sensitive cytogenetic marker in mutagen-exposed populations. The purpose of the present study is an extension of our earlier studies on unstable CA transmission through successive mitotic divisions. MATERIALS AND METHODS: The bromodeoxyuridine (BrdU) incorporation and fluorescence plus giemsa (FPG) method for M1-M5 metaphase analysis was carried out at 50, 72, 96 h to analyse TA and CA in conventionally and FPG stained chromosomes after irradiation of human blood samples with 3 Gy of gamma-rays. In situ hybridization (ISH) with enzymatic/fluorescence detection was used to analyse radiation-induced aneuploidy and TA. Analysis was carried out on sister chromatid exchanges (SCE) in M2 cells at 72 h and micronuclei (MN) at 24, 50, 72, 96 h. RESULTS: TA, corroborated by the absence of acentric fragments, were not detected in conventional/FPG stained/ISH chromosomes. Chromosome 21 aneuploidy was observed. Significant differences in mean frequencies of dicentrics/micronuclei (MN)/SCE with high frequency cells (HFC) were found in smokers after irradiation compared to non-smokers. Higher radiation induced CA in M1 cells were found with extended culture time. Induction of giant cells with mirror dicentrics, tricentrics and rings were found. CONCLUSION: TA in conventional or FPG stained metaphase chromosomes is not a sensitive cytogenetic marker for mutagen exposed population screening. Higher radiation induced CA frequencies in M1 cells with extended culture time were indicative of a delay in cell cycle progression of aberrant cells or different lymphocyte subset populations. Bridge-breakage-fusion (BBF) events due to dicentrics may be instrumental in the perpetuation of chromosomal instability. Differential effects were noted in radiation-induced dicentric, SCE and MN frequencies in smokers compared to non-smokers. Heavy smoking could be a confounding variable in chromosome-based biodosimetry and biomonitoring studies. Giant cells may denote a switch to amitotic modes of cell survival, providing additional mechanisms of genotoxic resistance.

Modulation of radiation-induced and mitomycin C-induced chromosome damage by apigenin in human lymphocytes in vitro.

Apigenin (APG), a flavone, is known to exhibit antioxidant, antimutagenic and antitumorigenic activity, both in vivo and in vitro. The aim of this study is to investigate the modulatory effects of APG on human lymphocytes after irradiation with gamma rays (3 Gy) or treatment with the antineoplastic agent, mitomycin C (MMC), in vitro. Cytogenetic biomarkers such as chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and cytochalasin-B blocked micronuclei (CBMN), were studied in blood lymphocytes treated with radiation, or antineoplastic agent (MMC), and APG. Whole blood lymphocytes were cultured in vitro using a standard protocol. No significant differences were found in the frequency of CAs or micronuclei (MN) in human peripheral blood lymphocytes irradiated with gamma rays (3 Gy) and then post-treated with APG. There was an increase in the frequency of SCEs per cell in APG-treated samples compared with the controls. Lymphocytes treated with MMC in the presence of APG exhibited a significant decrease (P < 0.01) in the frequency of SCEs compared with MMC treatment alone. The data for the MN test indicated that APG treatment significantly reduced (P < 0.01) the frequency of MMC-induced MN.

Characterization of Phlebotomus papatasi peritrophins, and the role of PpPer1 in Leishmania major survival in its natural vector.

The peritrophic matrix (PM) plays a key role in compartmentalization of the blood meal and as barrier to pathogens in many disease vectors. To establish an infection in sand flies, Leishmania must escape from the endoperitrophic space to prevent excretion with remnants of the blood meal digestion. In spite of the role played regarding Leishmania survival, little is known about sand fly PM molecular components and structural organization. We characterized three peritrophins (PpPer1, PpPer2, and PpPer3) from Phlebotomus papatasi. PpPer1 and PpPer2 display, respectively, four and one chitin-binding domains (CBDs). PpPer3 on the other hand has two CBDs, one mucin-like domain, and a putative domain with hallmarks of a CBD, but with changes in key amino acids. Temporal and spatial expression analyses show that PpPer1 is expressed specifically in the female midgut after blood feeding. PpPer2 and PpPer3 mRNAs were constitutively expressed in midgut and hindgut, with PpPer3 also being expressed in Malpighian tubules. PpPer2 was the only gene expressed in developmental stages. Interestingly, PpPer1 and PpPer3 expression are regulated by Le. major infection. Recombinant PpPer1, PpPer2 and PpPer3 were obtained and shown to display similar biochemical profiles as the native; we also show that PpPer1 and PpPer2 are able to bind chitin. Knockdown of PpPer1 led to a 44% reduction in protein, which in spite of producing an effect on the percentage of infected sand flies, resulted in a 39% increase of parasite load at 48 h. Our data suggest that PpPer1 is a component for the P. papatasi PM and likely involved in the PM role as barrier against Le. major infection.

Targeting the midgut secreted PpChit1 reduces Leishmania major development in its natural vector, the sand fly Phlebotomus papatasi.

BACKGROUND: During its developmental cycle within the sand fly vector, Leishmania must survive an early proteolytic attack, escape the peritrophic matrix, and then adhere to the midgut epithelia in order to prevent excretion with remnants of the blood meal. These three steps are critical for the establishment of an infection within the vector and are linked to interactions controlling species-specific vector competence. PpChit1 is a midgut-specific chitinase from Phlebotomus papatasi presumably involved in maturation and degradation of the peritrophic matrix. Sand fly midgut chitinases, such as PpChit1, whether acting independently or in a synergistic manner with Leishmania-secreted chitinase, possibly play a role in the Leishmania escape from the endoperitrophic space. Thus, we predicted that silencing of sand fly chitinase will lead to reduction or elimination of Leishmania within the gut of the sand fly vector. METHODOLOGY/PRINCIPAL FINDINGS: We used injection of dsRNA to induce knock down of PpChit1 transcripts (dsPpChit1) and assessed the effect on protein levels post blood meal (PBM) and on Leishmania major development within P. papatasi. Injection of dsPpChit1 led to a significant reduction of PpChit1 transcripts from 24 hours to 96 hours PBM. More importantly, dsPpChit1 led to a significant reduction in protein levels and in the number of Le. major present in the midgut of infected P. papatasi following a infective blood meal. CONCLUSION/SIGNIFICANCE: Our data supports targeting PpChit1 as a potential transmission blocking vaccine candidate against leishmaniasis.

p-Nitrophenol degradation via 4-nitrocatechol in Burkholderia sp. SJ98 and cloning of some of the lower pathway genes.

Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.

Pot and field studies on bioremediation of p-nitrophenol contaminated soil using Arthrobacter protophormiae RKJ100.

Biodegradation of p-nitrophenol (PNP), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. Bioremediation of PNP-containing soil was first carried out in pots using immobilized and free cells of Arthrobacter protophormiae RKJ100 in order to ascertain the role of a suitable carrier material. Results showed that stability of the introduced strain was enhanced upon immobilization and that the rate of PNP depletion decreased with increasing depth of soil. Small-scale field studies (in one square meter plots) were then conducted in which PNP-contaminated soil from an agricultural field was bioaugmented with strain RKJ100 under natural environmental conditions. PNP was totally depleted in 5 days by immobilized cells, whereas free cells were able to deplete 75% of PNP in the same time period. The fate of the released strain as monitored by plate counts, hybridization studies, and real-time polymerase chain reaction revealed fairly stable population of the cells upon immobilization on corncob powder throughout the period of study.