Stability profiling and degradation products of dihydromyricetin in Dulbecco's modified eagle's medium.

Dihydromyricetin has shown many bioactivities in cell level. However, dihydromyricetin was found to be highly instable in cell culture medium DMEM. Here, the underlying degradation mechanism was investigated via UPLC-MS/MS analysis. Dihydromyricetin was mainly converted into its dimers and oxidized products. At lower temperature, dihydromyricetin in DMEM showed higher stability. Vitamin C increased the stability of dihydromyricetin in DMEM probably due to its high antioxidant potential.

Seed bio-priming with tri-species consortia of phosphate solubilizing rhizobacteria (PSR) and its effect on plant growth promotion.

Three potential rhizobacteria namely Burkholderia gladioli (MTCC 10216), Pseudomonas sp. (MTCC 9002) and Bacillus subtilis (MTCC 8528) procured from IMTECH, Chandigarh (India) were evaluated individually and as consortia for its phosphate (P) solubilizing ability and effect of growth of fenugreek (Trigonella foenum-graecum L.) and tomato (Lycopersicon esculentum L.). Phosphate solubilizing ability of these strains individually and as consortia was tested on Pikovskayas agar medium, Phosphate solubilizing agar medium and National Botanical Research Institute phosphate agar medium containing six different sources of insoluble inorganic phosphate such as tri-calcium phosphate (TCP), di-calcium phosphate (DCP), zinc phosphate (ZP), ferric phosphate (FP), sodium di-hydrogen phosphate (SP), and aluminum phosphate (AP), and two organic P such as calcium and sodium phytate. The maximum P solubilizing ability was recorded in consortium-4 having all three potential bacterial strains. Phosphate solubilization after 7th day of incubation was 37.9 mg/100 ml of TCP, 40.01 mg/100 ml of DCP, 15.79 mg/100 ml of FP, 43.02 mg/100 ml of SP, no solubilization of ZP and AP, 39.75 mg/100 ml of calcium phytate and 24.01mg/100 ml of sodium phytate. Seed germination and the other plant parameters such as plant height and weight significantly increased in fenugreek and tomato seeds, bio-primed with consortium-4 followed by consortium-3. After bio-priming of seeds in pot assay, the level of phosphorus in soil got increased by 54% in consortium-4 treated soil followed by consortium-3 (47%) over untreated control soil. Based on these findings, consoritium-4 could be recommended as a good bio-inoculant for fenugreek, tomato and other crops in comparison to individual strains and other consortia.

A diterpenoid taxodone from Metasequoia glyptostroboides with antimycotic potential against clinical isolates of Candida species.

OBJECTIVE: The increasing importance of clinical isolates of Candida species and emerging resistance of Candida species to current synthetic antifungal agents have stimulated the search for safer and more effective alternative drugs from natural sources. This study was directed towards exploring the antimycotic potential of a diterpenoid compound taxodone isolated from Metasequoia glyptostroboides against pathogenic isolates of Candida species. MATERIALS AND METHODS: Antimycotic efficacy of taxodone was evaluated by disc diffusion assay, determination of minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations, and cell viability assay. To confirm a partial antimycotic mode of action of taxodone, the efficacy of taxodone was determined by measuring the release of 260 nm absorbing materials from the selected Candida species as compared to control. RESULTS: The taxodone at the concentration of 400 µg/disc displayed potential antimycotic effect against the tested clinical and pathogenic isolates of Candida species as diameters of zones of inhibitions, which were found in the range of 11 ± 0.0 to 12.6 ± 0.5mm. The MIC and MFC values of taxodone against the tested clinical isolates were found in the range of 250 to 1000 and 500 to 2000µ g/mL, respectively. On the other hand, the MIC and MFC values of positive control (amphotericin B) against the tested Candida isolates were found in the range of 62.5 to 250 and 500 to 2000 µg/mL. On the viable counts of the tested fungal isolates, the taxodone exerted significant antimycotic effect. Elaborative study of partial mode of action conducted onto the release of 260nm materials (DNA and RNA) revealed potential detrimental effect of taxodone on the membrane integrity of the tested pathogens at MIC concentration. CONCLUSION: With respect to the antimycotic effect of taxodone against pathogenic and clinical isolates of Candida species, it might be confirmed that bioactive compound taxodone present in M. glyptostroboides holds therapeutic value of medicinal significance.

Microbial fermentation of cabbage by a bacterial strain of Pectobacterium atrosepticum for the production of bioactive material against Candida species.

OBJECTIVE: The objective of this study was to produce the bioactive fermented product by the microbial fermentation of cabbage (Brassica oleracea) using a bacterial strain Pectobacterium atrosepticum which was assessed for its antimycotic efficacy against pathogenic isolates of Candida species. MATERIALS AND METHODS: An approach of microbial fermentation of cabbage using a bacterial strain P. atrosepticum was applied to obtain the bioactive fermented product. Antimycotic efficacy of bioactive fermented product of cabbage was evaluated by disc diffusion assay, minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations, cell viability assay and scanning electron microscopy (SEM) analysis. RESULTS: The bioactive fermented product (500µg/disc) revealed promising antimycotic effect against the tested Candida species as a diameter of inhibition zones (10±0.2 to 13±0.4mm) along with its MIC and MFC values, ranging from 250 to 1000 and 250 to 2000µg/ml, respectively. Exposure of 140min of bioactive fermented product exerted potential antimycotic effect on the viable counts of the tested fungal isolates with about 85 to 100% inhibitory effect. Further, the study of SEM revealed potential detrimental effect of bioactive product on the morphology of C. albicans KACC 30003 at MIC concentration. Elaborative study of GC-MS analysis conducted on bioactive fermented product of cabbage revealed transformation products present in fermented product. CONCLUSION: These results confirmed the therapeutic potential of microbially bioconverted/fermented products of cabbage for using in medicinal and pharmaceutical preparations.

In vitro inhibition of food spoilage and foodborne pathogenic bacteria by essential oil and leaf extracts of Magnolia liliflora Desr.

The aim of this study was to examine the chemical composition of the essential oil isolated from the floral parts of Magnolia liliflora Desr. by hydrodistillation, and to test the efficacy of essential oil and various leaf extracts against a diverse range of microorganisms comprising food spoilage and foodborne pathogenic bacteria. The chemical composition of essential oil was analyzed by GC-MS. It was determined that 52 compounds, which represented 78.07% of total oil, were present in the oil. The oil contained mainly levoxine (15.59%), methylcyclopropane (24.26%), 2-beta-pinene (5.3%), caryophyllene oxide (4.01%), and beta-caryophyllene (1.7%). The oil (1000 ppm/disc) and leaf extracts (1500 ppm/disc) exhibited promising antibacterial effects against the tested pathogens as a diameter of zones of inhibition (9 to 18 and 7 to 12 mm) and MIC values (125 to 1000 and 500 to 3000 microg/mL), respectively. Also, the oil had a potent detrimental effect on the viable count of the tested bacteria. The results obtained in this study support the role of essential oil and the leaf extracts derived from M. liliflora as a remarkable approach to inhibit the growth of food spoilage and foodborne pathogens.

Novel disulphide esters of carbothioic acid as potent, non-detergent spermicides with low toxicity to Lactobacillus and HeLa cells in vitro.

BACKGROUND: The design, synthesis, characterization and evaluation of a novel series of non-detergent spermicides has led to the discovery of two unique molecules (DSE-36 and DSE-37) that were approximately 25 times more potent spermicides than nonoxynol-9 (N-9). METHODS: Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer Assay), the effect on sperm-membrane integrity [hypo-osmotic swelling test (HOST)], supravital staining and scanning electron microscopy (SEM) and the induction of apoptosis [fluorescein isothiocyanate (FITC) Annexin-V and JC-1 labelling using flow cytometry] by the new class of compounds. HeLa and Lactobacillus cultures were used to assess the cytotoxicity of compounds and their compatibility to normal vaginal flora, respectively. RESULTS: Compounds DSE-36 and DSE-37 exhibited a strong spermicidal activity [minimum effective concentration (MEC) = 0.002%], which was approximately 25 times more potent than that of N-9 and Sapindus saponins (MEC = 0.05%). As compared with surfactants, DSE-36 and DSE-37 were found to be safer at MEC towards the growth and survival of Lactobacilli and HeLa cells in vitro and to have a milder effect on sperm plasma membrane. At EC(50) both induced apoptosis in sperm cells as characterized by increased labelling with Annexin-V and decreased polarization of sperm mitochondria. CONCLUSION: Preliminary studies have revealed that in sharp contrast to the non-specific surfactant action of N-9, DSE-36 and DSE-37 have a highly potent, mechanism-based, detrimental action on human sperm. The unique ability of these non-detergent molecules to selectively kill sperm and spare Lactobacilli and HeLa cells at MEC values much lower than that required for N-9 indicates their potential as superior ingredients for formulation into microbicidal contraceptives.

Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.

Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin-1, a prominent NOS-interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L-[3H] arginine and 4,5-diaminofluorescein diacetate (DAF-2DA). Reverse transcriptase-polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L-[3H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin-1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin-1 in rat PMNs.

Argemone oil induced cellular damage in the reproductive tissues of transgenic Drosophila melanogaster: protective role of 70 kDa heat shock protein.

We explored the reproductive toxicity of argemone oil and its principal alkaloid fraction in transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9). The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Freshly eclosed first instar larvae of transgenic Drosophila melanogaster were transferred to different concentrations of argemone oil and its alkaloid fraction contaminated food. Virgin flies that eclosed from the contaminated food were pair-mated to look into the effect on reproduction. The study was further extended by investigating hsp70 expression and tissue damage in larval gonads, genital discs, and reproductive organs of adult fly. Our results showed that argemone oil was more cytotoxic than its principal alkaloid fraction. Moreover, it was the male fly that was more affected compared to its opposite number. The accessory glands of male reproductive system of the fly, which did not express hsp70, exhibited severe damage as evidenced by Trypan blue staining. This prompted us to explore the ultrastructural morphology of the gland, which showed acute signs of necrosis in both the cell types as evident by necrotic nuclei, higher vacuolization, and disorganized endoplasmic reticulum, decrease in the number of Golgi vesicles and disorganized, loosely packed filamentous structures in the lumen of the accessory gland, at the higher concentrations of the adulterant. The study showed the reproductive toxicity of argemone oil and its alkaloid fraction in transgenic Drosophila melanogaster and further confirmed the cytoprotective role of hsp70.

Histological and ultrastructural regulation in rabbit endometrial explants by estrogen in serum-free culture.

A repertoire of hormonal signals including estrogen regulate the growth, differentiation, and functioning of diverse target tissues, including the ovary, the mammary gland, and skeletal tissue. A serum-free culture system derived from rabbit endometrium explants has been devised and is reported here to explore estrogen action in vitro. The system involves aseptically harvesting the uterus from a virgin rabbit, dissecting the endometrium, explanting it into 1- to 2-mm(3) pieces weighing approximately 1-2 mg each, and incubating these pieces in serum-free Medium-199. The culture is carried out for a period of 4 d in a humidified CO(2) incubator at 37 degrees C with 5% CO(2). The effect of extraneously added estrogen (1 microg/ml) was investigated by histological and ultrastructural procedures. It was observed that estrogen could induce specific changes, such as abundant mitochondria, rough endoplasmic reticulum, golgi complex, and intracellular collagen deposition, in both the epithelial and the fibroblast cell components of the explanted tissue. The study, therefore, indicates that the proposed system is an ideal tool for exploring and demonstrating estrogen responsiveness under in vitro conditions.

Stromal cell interaction and relevance to predecidual events and menstruation.

Endometrial stromal cells play a vital role during decidualization and implantation. The aim of this study was to analyse the cyclic ultrastructural variations of stromal fibroblasts, granulocytes and blood-derived cells which invade the stroma during various stages of the menstrual cycle. Diverse opinions exist in the literature regarding the origin, fate and function of the endometrial granulocytes. Our study is in agreement with available immunological data and shows that (i) fibroblasts and granulocytes are distinct cell types in the stroma, (ii) they exhibit distinct changes across the menstrual cycle and (iii) fibroblasts are not the common progenitor for granulocytes and predecidual cells in the secretory phase endometrium, as suggested by previous investigators. The infiltration of eosinophils, platelets, macrophages, neutrophils, etc., during the menstrual phase makes the stroma a potential source of growth factors and cytokines which may regulate the process of regression and regeneration of the endometrium. Furthermore, we propose that endometrial granulocytes could be the source of decidual prolactin because the ultrastructural morphology of their secretory granules closely resembles that of the prolactin-secreting cells in the pituitary.

Cyclic alterations in rhesus monkey endometrium by scanning electron microscopy.

The present study was undertaken to correlate the surface topography of endometrium with altered concentrations of circulating steroids during different phases of the artificially-induced menstrual cycle. Scanning electron microscopy demonstrated that oestradiol during the oestrogenic phase induces an increase in the number of ciliated cells and the size of microvilli; by contrast, progesterone, in addition to inducing secretory activity in non-ciliated cells, had a negative effect on oestradiol-dependent morphological characteristics of ciliated cells, causing a reduction in cell number and deciliation, and inducing drooping of cilia during the progestogenic phase of the artificially-induced menstrual cycle. In addition, surface and glandular epithelial cells both actively participate in restoration of the endometrial surface during the menstrual phase, an event which parallels endometrial shedding.

Platelets in essential hypertension.

The ultrastructure and adenosine diphosphate induced aggregatory response of platelets of 39 patients with essential hypertension have been studied. Both, transmission and scanning electron microscopy showed the co-existence of various morphological forms of platelets suggestive of activation. The hypertensive platelets also exhibited increased sensitivity to ADP induced aggregation confirming the activated state of platelets; the degree of activation correlated well with the levels of diastolic blood pressure.

Platelet ultrastructural morphology and its relevance in essential hypertension.

A preliminary study of platelet ultrastructural morphology by transmission electron microscopy in patients with established essential hypertension was carried out. Platelets of hypertensive patients were found to be in various stages of 'platelet activation'. According to the degree of transformation observed, platelets were classified into six forms: discoid, pseudotubular, membranous, saccular, pseudopodical and hyaline and correlated to the severity of hypertension. The pseudopodical and hyaline types platelets were particularly observed in severely hypertensive patients, and/or those with clinical evidence of target organ damage. Platelet ultrastructural changes may form the basis of a better understanding of the aetiology and pathogenesis of essential hypertension, and may serve as a marker in assessing the extent of underlying in vivo endothelial injury and tendency to thrombotic complications.

Effect of conditioning the enamel surface of primary teeth with citric acid: a SEM study.

The present SEM study revealed the effect of 50 percent citric acid on the primary tooth enamel subsequent to mechanical pretreatment. The enamel of primary teeth was conditioned with 50% citric acid for 1, 1.5, 2, 3 and 5 minutes. A uniformly etched enamel surface was obtained at 5 minutes.

Changes in uterine surface epithelium of rat during pregnancy.

The first site of attachment of the trophoblast to the uterus in rodents is on the antimesometrial side and the inner cell mass is oriented mesometrially. The present study was performed with a view to study if comparable changes occur on both the mesometrial and antimesometrial side during early pregnancy in the rat using the scanning electron microscope. The results showed three types of ectoplasmic projections: namely, (i) a smooth surface and a narrow base; (ii) many folds and pits; and (iii) a ruffled surface and broad base, seen in clusters and located at intervals on the antimesometrial side only. The results suggest that these projections with ruffled surfaces may play a role in adhesion of the blastocyst and localisation of implantation sites in the rat.

Surface ultra-architecture of supraependymal elements in the vicinity of the area postrema of rhesus monkeys.

The caudal part of the fourth ventricle in adult rhesus monkeys, of both sexes, revealed the area postrema (AP) to be composed of bilateral spindle-shaped elevations located ventromedial to the gracile tubercles. Supraependymal (SE) elements, comprised of SE cells and/or fibre processes could be seen near the medial margin of a narrow oligociliated strip separating the nonciliated AP from the profusely ciliated vagal triangle (VT). In general, these consisted of multilobed ganglion-like cell clusters interconnected by fasciculated fibre processes with varicosities along their lengths. Thinner processes radiating from the cell clusters and varicosities traversed towards the VT for short distances and often appeared to penetrate the underlying ependyma. The thicker fibre processes which exhibited a fasciculated configuration extended between raised nodule-like areas in the ventricular floor. The majority of such areas exhibited an uneven surface due to the presence of numerous membranous folds over them. Near the periphery of nodular areas as well as along the fasciculated fibre processes interconnecting them, a few bouton-like protrusions were discernible. The SE elements observed in the caudal portion of the fourth ventricular floor are suggestive of comprising some integrative pathways between the ventricular cerebrospinal fluid and subependymal functional centres in the vicinity of the AP, a circumventricular organ having diverse functions.

Morphological changes in human spermatozoa as examined under scanning electron microscope after in vitro exposure to saponins isolated from Sapindus mukorossi.

Saponins isolated from Sapindus mukorossi have potent spermicidal activity. Morphological changes in human ejaculated spermatozoa after exposure to these saponins were evaluated under Scanning Electron Microscope. The minimum effective concentration (0.05% in spot test) did not affect the surface topography after exposure for 1 minute. However, incubation of spermatozoa for 10 minutes resulted in extensive vesiculation and disruption of plasma membrane in the head region. Higher concentrations (0.1%, 1.25%, 2.5% and 5.0%) caused more or less similar changes which included vesiculation, vacuolation, disruption or erosion of membranes in the head region. These findings suggest that the morphological changes observed are due to alterations in the glycoproteins associated with the lipid bilayer of plasma membrane of spermatozoa.

Effects of sodium deoxycholate on the mechanical and electrical activities and ultrastructure of guinea pig atria.

The mechanism of cardio-inhibitory effects of sodium deoxycholate (DOC) was investigated by studying its effects on the contractility, action potentials (APs) and ultrastructure of guinea pig atrial preparations. DOC (10(-7)-10(-4) M) caused reversible negative ino- (NIE) and chrono-tropy in spontaneously beating (SBA) and NIE in electrically driven left (EDA) atria. At higher doses (greater than or equal to 1.10(-3) M) DOC caused irreversible inhibition of contractions. Atropine (10(-7)-10(-4) M) failed to inhibit both the reversible and irreversible effects of DOC. The NIE due to lower doses of DOC (less than or equal to 1.10(-4) M) was inhibited by higher [Ca2+]0, isoprenaline (10(-6)-10(-4) M), and noradrenaline (10(-6)-10(-5) M), which did not alter the dose of DOC required for the irreversible and complete NIE. In lower doses (10(-7)-10(-4) M) DOC caused a reversible inhibition of the AP durations at -20 and -40 mV (APD20 and APD40, respectively), but increased the AP duration at 90% repolarisation (APD90). At higher doses (greater than 5.10(-4) M) it caused an irreversible membrane depolarization, reduction in APD20 and APD40, and complete cessation of electrical activity. The ultrastructural changes in atria treated with 1.10(-4) M DOC were characterized by poorly delineated glycocalyx and at greater than 1.10(-3) M by disruption of sarcolemma and sarcoplasmic reticulum and swelling disruption of mitochondria. Taken together these observations show that DOC caused reversible and irreversible inhibition of atrial contractions at low (10(-7)-10(-4) M) and high (greater than 5.10(-4) M) concentrations, respectively, by different mechanisms. The former effect is due to inhibition of Ca2+ channel activity and the latter due to its detergent property causing removal of subcellular components.