Effect of salicylic acid and silver nitrate on rutin production by Hyptis marrubioides cultured in vitro

ABSTRACT: Hyptis marrubioides (Lamiaceae) is a medicinal plant that is native from Brazilian Cerrado. In vitro propagation techniques make use of elicitors to alter metabolic pathways, affecting how molecules are produced both qualitatively and quantitatively. This research aimed to evaluate how abiotic elicitors salicylic acid (SA) and silver nitrate (SN) at concentrations of 30µM or 60µM influence Hyptis marrubioides seedling growth by two different in vitro culture methods. The rutin content was quantified by HPLC-DAD. Compared to an untreated culture, the H. marrubioides methanolic extracts cultured in MS medium for 10 days followed by culture in MS medium containing SN (30µM) for 20 days had 1.28 times higher rutin content. In a second experiment, seedlings were cultured in MS medium for 20 days, and then the desired elicitor was added to the culture and allowed to remain in contact with the medium for three and six days. SA (30µM) gave the best results: rutin production was 16.56-foldhigher than the control after six days. SN (30µM) increased the rutin content by 1.17-fold. At the two concentrations evaluated during the elicitation experiments, neither SA nor SN altered the growth parameters shoot length, leaf number, and fresh and dry weight of H. marrubioides seedlings grown in vitro as compared to the control. Based on these results, the abiotic elicitors SA and SN successfully provide Hyptis marrubioides with increased rutin content in vitro.

Growth of Candida sp and Aspergillus sp from Bronchoscopy Pulmonary Tuberculosis Patients on Bran Media

Pulmonary tuberculosis caused by Mycobacterium tuberculosis become ahealth problem in Indonesia. The chronic nature of this disease is further exacerbated ifit is accompanied by fungal infection such as Candida albicans and Aspergillus sp.,which is usually remains undiagnosed and thus untreated. Culture techniques can beused to identify Candida sp and Aspergillus sp from bronchoscopy. Fungal culturemedia in laboratory containing high carbohydrate source, nitrogen source are requiredfor the growth. This nutrient can be found in bran that contains high carbohydrates,proteins, fats, vitamins, and crude fiber. So that bran can be used as raw material for alternative fungal growth media. The purpose of this study was to increase bran as amedium for the growth of Candida sp and Aspergillus sp isolated from bronchoscopy ofpulmonary TB patients. This study included bran collection, preparation of bran media,inoculation bronchoscopy on bran media, observation of fungal growth. Colonies ofCandida sp and Aspergillus sp were confirmed microscopically. The results showed thatCandida sp and Aspergillus sp grew on both media, Bekatul Dextrose Agar and PotatoDextrose Agar. The conclusion of this study is that bran can be used as a medium forfungal growth. Bran media can be used as an alternative media to replace syntheticmedia to grow Candida sp and Aspergillus sp isolated from bronchial rinses.

Growth Inhibition of Human Multiple Myeloma Cells by a CD40 Ligand (CD40L, CD154) Transgene - Oncolytic Virus Construct.

Human multiple myeloma (MM) remains incurable despite recent advances in induction therapy. To explore biotherapeutic approaches that may potentially enhance the efficacy of conventional treatment, we examined the growth regulatory properties of CD40L (CD154), the natural ligand for the myeloma cell surface receptor, CD40. Based on our previous findings that the recombinant CD40L protein effectively inhibited human MM cell growth, a conditional-replicative adenoviral construct, AdEHCD40L was used for targeted delivery of the CD40L transgene. AdEHCD40L incorporates tumor/tissue specific promoters that limit viral and transgene expression to HIF (hypoxia inducing factor)-1α overexpressing cells, which are prevalent in the human bone marrow compartment. Conditional expression of the early adenoviral E1A gene and the CD154 transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.68% and 30%, respectively). Further, treatment with AdEHCD40L at a multiplicity of infection (MOI) of 1 resulted in pronounced growth inhibition for both cell lines (95.5±2.1% and 80.5±9.8%, mean±SD, respectively). AdEHCD40L treatment was more effective than the parental construct without the CD154 transgene (AdEHNULL) in both cell lines (p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells (0% at 48hrs) and normal fibroblast cells IMR-90 (2.8±0.3%). The in vivo antitumor activity of AdEHCD40L was examined with a subcutaneous RPMI 8226 heterotransplant model in SCID mice. Intratumoral injection of AdEHCD40L (5x107 pfu, x 5) reduced xenograft growth by 53% at day 29 (4.8±0.9 mm, vs. 10.5±1.2 mm in mock-treated animals; p=0.002), and was more effective than AdEHNULL (7.6±1.1 mm; p=0.03). Adenoviral hexon and CD40L expression was detectable at 29 days post-viral treatment, based on immunohistochemical analysis. Hence intratumoral treatment with AdEHCD40L likely involved oncolytic viral replication. To further characterize cellular events that accompany MM cell growth inhibition, apoptotic activity was measured by annexin V and propidium iodide incorporation. A marked elevation of the annexin V+ subset (21.3±6.5%, vs. 7.7±1.9% in untreated culture; p=0.007) was accompanied by decreased cell viability (52.8±1%, vs. 76.1±11% in untreated cultures; p = 0.04) following AdEHCD40L treatment. Cell cycle distribution analysis demonstrated a corresponding increase in the subG0/G1 compartment (AdEHCD40L, 23.9±3.6%; vs. untreated, 9.1±2.3%) that was consistent with elevated apoptosis. Further, AdEHCD40L increased S phase accumulation by 72 hrs (68.0±2.4%, vs. 54.0±5.5% in AdEHNULL and 48.9±4.6% in untreated culture; p<0.003). Currently, gene expression array analyses are underway to define molecular events that are pertubated by CD154 transgene activity and by viral oncolysis. These findings will further elucidate the mechanism of action of the CD154+oncolytic viral approach for experimental gene therapy of human MM.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

Penciclovir is a selective inhibitor of hepatitis B virus replication in cultured human hepatoblastoma cells.

Penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yI)guanine], an effective antiherpesvirus agent, was found to be a potent and selective antiviral agent against intracellular hepatitis B virus (HBV) replication (drug concentration at which a 10-fold decrease in HBV DNA from the average level in an untreated culture was observed [EC90], 1.6 microM) and extracellular virion release (EC90, 0.7 microM) by cultured human hepatoblastoma (2.2.15) cells. Acyclovir and three other related 9-alkoxypurines with activity against either herpesviruses or human immunodeficiency virus were uniformly inactive against HBV. The activity of penciclovir is discussed in relation to recent findings related to its mode of action against HBV.

Spore-forming bacteria that carboxylate phenol to benzoic acid under anaerobic conditions

A methanogenic consortium transforming phenol to benzoic acid was submitted to different treatments to characterize the carboxylating microorganisms and eventually to facilitate their isolation. Under aerobic conditions, phenol was not transformed by the consortium and no growth was observed on solid medium. The consortium from an inoculum that was treated with heat, or heat and ethanol, retained the ability to carboxylate phenol under strictly anaerobic conditions. Electron microscopic observations of the consortium from an inoculum that was heated for 15 min at 80 °C revealed only Gram-positive bacilli. In this culture, methane production was not detected and benzoic acid accumulated. Five colonies with distinct morphologies were isolated from this culture on solid medium. Four of these strains were identified as Clostridium spp. In contrast to the untreated culture, none of the strains isolated were able to carboxylate phenol in pure culture or in coculture, nor could they decarboxylate or dehydroxylate 4-hydroxybenzoic acid, or oxidize 2-hydroxybenzyl alcohol, or O-demethylate anisole or 2-methoxyphenol. Also, the consortium from a treated inoculum retained its ability to decarboxylate and dehydroxylate 4-hydroxybenzoic acid forming phenol and benzoic acid, respectively, but could not accomplish the other reactions. These results suggest that spore-forming microorganisms are involved in the carboxylation of phenol and in the decarboxylation and dehydroxylation of 4-hydroxybenzoic acid.Key words: spore-forming bacteria, phenol, benzoic acid, methanogenic conditions, carboxylation.

Growth and nucleic acid synthesis of Salmonella typhimurium inhibited by α-methylmethionine

The addition of α-methylmethionine to exponentially growing cultures of Salmonella typhimurium or Escherichia coli resulted in an immediate cessation of growth with low concentrations of the analogue. After a lag period, the length of which was dependent on the optical density of the culture, growth resumed at a rate characteristically slower than that of an untreated culture. Chemical analyses showed that the rate of synthesis of ribonucleic acid and protein by S. typhimurium had a pattern similar to the curve for optical density. However, deoxyribonucleic acid and cell numbers continued to increase at the normal rate for about 30 minutes, then slowed, and changed to the rate characteristic of cells treated with the analogue. Incorporation of thymine-14C, uracil-14C, and leucine-14C by the thymine auxotroph E. coli 15T− confirmed the chemical analyses. After 25 minutes the cells of the culture treated with α-methylmethionine had incorporated only 15% of the uracil and 26% of the leucine as that of a control culture. The optical density was 30% that of a control culture. Thymine incorporation was 92% that of a control culture.

The Action of Acriflavine upon Bodo caudatus

1. A study has been made of the effect of acriflavine upon Bodo caudatus. The drug has the property of producing a percentage of modified Bodos without parabasal bodies but no permanently aparabasal strains have been produced.2. A technique was devised whereby the resistance, as judged by two different types of survival experiment, could be correlated with the percentage of modified Bodos produced when the strain is put to grow in an ascending series of concentrations of acriflavine incorporated in the culture medium.3. The peak of the count of altered Bodos in relation to the concentration in which it occurs is found to be the index of the sensitiveness of the strain at that date. The maximum reaction as gauged by the numbers of modified Bodos produced in an untreated strain of average sensitiveness is about 70 to 80 per cent., and this occurs in so low a concentration as 1/1,000,000. 1/5,000,000 gives a count below the maximum and 1/50,000,000 produces only 1 to 4 per cent. according to the sensitiveness of the strain.4. The mass untreated strain was originally derived from a single Bodo isolated in the autumn of 1926. This strain in 1927–28 was used as the mass untreated strain in the experiments. Its resistance was found to fluctuate from below a capacity to evolve with great difficulty in 1/50,000 to a somewhat feeble development in 1/10,000 acriflavine incorporated plates. The mass untreated strain was never able to evolve in plates containing 1/5000 acriflavine at any time.5. Strains grown from single isolations from the untreated mass culture showed different degrees of natural resistance. The most resistant clones could develop in 1/5000 acriflavine incorporated plates, but not in 1/2000. The great importance of this natural range of resistance in estimating an acquired drug fastness is emphasized. Strains which had been made resistant could be brought to live continuously in 1/1500 acriflavine incorporated plates, but no evolution was obtained in 1/1000.6. Evidence that drug fastness in Bodo caudatus is due to the interaction of selective inheritance and the actual modification of the quality of the Bodo by evolution in the drug is given. The modification is not apparently a mutation, it is a heritable piling up of changes in a particular direction.7. A high resistance once acquired is retained through prolonged cultivation upon drug-free media. A gradual loss occurs, partly by a dilution through multiplication of the character impressed and partly by the survival of variants of less resistance to acriflavine, but of perfect viability in other circumstances and the competition of these within the strain. The loss of resistance is greatly accelerated in single cell cultures (clones) isolated from the resistant strains, but up to the present no treated strain has entirely lost the effect of the original exposures to the drug. The longest time elapsing between the creating of a partially resistant strain and its test for resistance above that of the untreated culture is one year.8. The bearing of the data obtained in Bodo caudatus upon the condition found in trypanosomiasis and upon certain aspects of chemotherapy in this group is discussed.9. Bodo caudatus being an organism without conjugation and therefore without bi-parental inheritance and consequently lacking the reorganising effect that such a periodic closing of the cycle produces, affords an example of a labile organism capable of being progressively altered within certain limits under the influence of the environment.