Lysis of growing cells ofSaccharomyces cerevisiae induced by papulacandin B

Light and electron microscopy was used to study the effect of papulacandin B onSaccharomyces cerevisiae in the exponential growth phase. At 1–2 μg/mL cell division in the culture continued almost in parallel with the control, at 4 μg/mL cell proliferation was reduced and the culture contained some cells with 2–9 buds which were not separated from the mother cell by a septum, and at higher concentrations (8, 16 and 32 μg/mL) the proliferation stopped within 2 h. Cessation of proliferation was due to lysis of budding cells in the bud region including perforation of thinned cell wall (most often at the bud basis and sometimes at its apex), extrusion of cytoplasm and death of cell. Lysis was also observed in cells without visible buds. Dividing cells died without visible lysis.     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Differential Effects of selenium on the proliferation of human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro

The effect of different concentrations of Na₂SeO₃ on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 μg/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1–5μg/mL sodium selenite for 1–3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 μg/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei.     

Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos

Summary Somatic proembryos of mango (Mangifera indica L. cv. Hindi) were co-cultivated withAgrobacterium tumefaciens strain A208 harboring pTiT37-Se::pMON 9749 (9749 ASE). Transformed somatic proembryos capable of growing on selection medium containing 200 μg/ml kanamycin produced the characteristic indigo blue precipitate in the presence of 5-bromo-4-chloro-3-glucuronic acid. These proembryos were chimeral consisting of transformed (blue) and nontransformed (yellow/white) cells. A stepwise selection strategy was found necessary to eliminate chimeras. a) Initial screening at 200 μg/ml kanamycin to enable growth of transformed cells, b) further screening at 400 μg/ml kanamycin to reduce chimeras, and c) recovery of pure transformed clones of proembryos in liquid selection medium with 100 μg/ml kanamycin. The integration of the NPT II and GUS genes into mango genome was confirmed by Southern hybridization.     

Influence of Organic Buffers on Bacteriocin Production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ST110

The effect of the organic buffer salts MES, MOPS, and PIPES on the growth of S. thermophilus ST110, medium pH, and accumulation of the antipediococcal bacteriocin thermophilin 110 were evaluated in whey permeate media over a period of 24 h. In nonbuffered medium, thermophilin 110 production at 37°C paralleled the growth of S. thermophilus ST110 and reached a maximum after 8–10 h. Addition of organic buffer salts decreased the drop in medium pH and resulted in increased biomass (dry cells; μg/mL) and higher yields of thermophilin 110 (units/μg cells). The best results were obtained by the addition of 1% (w/v) MES to the medium, which reduced the pH drop to 1.8 units after 10 h of growth (compared to 2.3 pH units in the control) and resulted in a 1.5-fold increase in cell mass (495 μg/mL) and a 7-fold increase in thermophilin 110 yield (77 units/μg dry cells) over the control. The results showed that whey permeate-based media may be suitable for producing large amounts of thermophilin 110 needed for controlling spoilage pediococci in industrial wine and beer fermentations. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Changes of Sodium Iodide Symporter Regulated by IGF-I and TGF-β1 in Mammary Gland Cells from Lactating Mice at Different Iodine Levels

The sodium/iodide symporter (SLC5A5, also known as NIS) is a transmembrane glycoprotein. Physiologically, iodide transportation in the mammary gland occurs during late pregnancy and lactation. To identify factors that may regulate this process at different iodine levels, we have studied the expression of NIS gene and protein in cultured mammary gland explants from lactating mice by real-time quantitative PCR and In-Cell Western methods. Mammary gland cells were grown in media with different levels of iodine for 24 h. The iodine treatment groups consist of low iodine group I (LI-I, 0 μg/l), low iodine group II (LI-II, 5 μg/l), control group (C, 50 μg/l), high iodine group I (HI-I, 3,000 μg/l), and high iodine group II (HI-II, 10,000 μg/l). The cells were then incubated with or without insulin-like growth factor I (IGF-I) or transforming growth factor β1 (TGF-β1) for another 24 h. We found that iodine inhibited NIS mRNA and protein expression in a dose-dependent manner. IGF-I and TGF-β1 further decreased NIS mRNA and protein expression that iodine inhibited at different iodine levels. In summary, we have shown that iodine downregulated NIS expression in cultured mammary gland explants from the lactating mouse. IGF-I and TGF-β1 inhibited NIS mRNA and protein expression in the mammary gland under different iodine levels.     

Study of the Action of Se and Cu on the Growth Metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Microcalorimetry

The biological effect of Se and Cu²⁺ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu²⁺ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²⁺ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu²⁺ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu²⁺ on E. coli when Se was higher than 10 μg/ml and Cu²⁺ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu²⁺ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen.     

Definitions and Epidemiology of Candida Species not Susceptible to Echinocandins

Antifungal susceptibility testing of Candida against the echinocandin antifungal agents (anidulafungin [ANF], caspofungin [CSF], micafungin [MCF]) has been standardized by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Testing. The CLSI proposed a single set of clinical breakpoints (CBPs) for all three echinocandins and all species of Candida: susceptible, minimum inhibitory concentration (MIC) ≤ 2 μg/mL; nonsusceptible, MIC > 2 μg/mL. Subsequently, these CBPs have been shown to lack sensitivity in detecting strains of Candida with acquired resistance mechanisms associated with treatment failure. Studies using the CLSI method have defined wild-type (WT) MIC distributions and epidemiologic cutoff values (ECVs) for each echinocandin and the common species of Candida. The ECVs serve as a sensitive means of discriminating WT strains from those with acquired resistance mechanisms. WT MIC distributions revealed ECV ranges of 0.03 to 0.25 μg/mL for all major species except C. parapsilosis (1–4 μg/mL) and C. guilliermondii (4–16 μg/mL). These ECVs reliably differentiate WT strains of each species from non-WT strains containing fks mutations. These data, coupled with additional biochemical, clinical, pharmacokinetic, and pharmacodynamic considerations, have resulted in new CBPs of ≤0.25 μg/mL (susceptible), 0.5 μg/mL (intermediate), and ≥1 μg/mL (resistant) for ANF, CSF, and MCF for C. albicans, C. tropicalis, and C. krusei. For these agents and C. parapsilosis, the new CBPs are ≤2 μg/mL (susceptible), 4 μg/mL (intermediate), and ≥8 μg/mL (resistant). For C. glabrata, the CBPs for ANF and CSF are ≤0.12 μg/mL (susceptible), 0.25 μg/mL (intermediate), and ≥0.5 μg/mL (resistant), whereas those for MCF are ≤0.06 μg/mL, 0.12 μg/mL, and ≥0.25 μg/mL, respectively. Application of both ECVs and the lower species-specific CBPs for the echinocandins has proven useful in both resistance surveillance and clinical care and will serve as an important step in international harmonization of in vitro susceptibility testing of this important antifungal class.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

Tyrosine kinase activation in LPS stimulated rat kupffer cells

Kupffer cells, a majority of the body's fixed macrophages, are a major site of bacterial lipopolysaccharide (LPS) metabolism and are mediators in the body's response to sepsis. Uptake of LPS is different in Kupffer cells than other macrophages. Signal transduction in other macrophages in response to LPS involves phosphorylation of proteins in the 50–60 kDa range. We hypothesized that Kupffer cells may have unique signal transduction pathways in response to LPS. Rat Kupffer cells were exposed to LPS (1 μg/mL) for varying times ranging from 15 to 90 min. Cell lysates were Western blotted using an anti-phosphotyrosine antibody. The blots showed an increase in the amount of tyrosine phosphorylation on two proteins of 119 kDa and 83 kDa. The effects of varying LPS concentration (1 ng/mL-1 μg/mL) showed an increasing amount of phosphorylation with increasing LPS concentration. To associate the importance of tyrosine phosphorylation in the response of Kupffer cells to LPS, the tyrosine kinase inhibitors, tyrphostin, lavendustin, and genisten were used to study the effects of inhibiting phosphorylation on TNF-α production. Kupffer cells were preincubated in the presence of the inhibitor and exposed to LPS (1 μg/mL). TNF-α was measured in the conditioned media by ELISA. A 70% or greater decrease in TNF-α production was observed. When phagocytosis of latex beads by rat Kupffer cells was measured in vivo using intravital video microscopy, LPS treatment significantly increased uptake. This increase in phagocytosis was inhibited by tyrphostin. These results show what may be unique phosphorylation events in Kupffer cells that are related to LPS induced production of TNF-α. Presented in part at the American Association for the Study of Liver Diseases Annual Meeting, Chicago, IL (USA), November 3–7, 1995.     

Susceptibility of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains toward combinations of oxacillin-2,4-dihydroxychalcone

In order to determine the existence of synergism of the bacteriostatic action of flavonoids against G⁺ bacteria between a clinically interesting conventional antibiotic and a flavonoid, combinations of oxacillin (OXC) and 2,4-dihydroxychalcone (DCH) as enhancer were assayed against methicillin-sensitive Staphylococcus aureus ATCC 29 213 and methicillin-resistant S. aureus ATCC 43 300. Using a kinetic-turbidimetric method, growth kinetics was monitored in a broth containing variable amounts of OXC alone and combinations of variable OXC-constant DCH. The minimum inhibitory concentrations (MIC) of OXC alone and in combination with DCH were evaluated. For the 29 213 strain, OXC MIC was 25 μg/mL, while combinations of 2–8 μg/mL OXC with 10 μg/mL of DCH totally inhibited growth and showed synergism. The resistance of the 43 300 strain in the presence of OXC was verified; OXC-DCH combinations decreased bacterial growth by 35 %. DCH augments the action of OXC against methicillin-susceptible S. aureus and therefore constitutes a good bacteriostatic agent for methicillin-resistant S. aureus.     

Sensitivity of selected members of the family Halobacteriaceae to quinolone antimicrobial compounds

Many members of the Halobacteriaceae are inhibited by quinolone compounds, which inhibit type II DNA topoisomerase. Ciprofloxacin was the most potent inhibitor, followed by ofloxacin and norfloxacin. Ciprofloxacin concentrations between 25 and 60 μg/ml caused 50% inhibition of the growth of most Haloferax and Haloarcula species. Halobacterium species were less sensitive. At sublethal concentrations, formation of elongated and/or swollen cells was observed in many species. The alkaliphilic Natronobacterium pharaonis was very sensitive (50% inhibition by ciprofloxacin, ofloxacin, and norfloxacin at concentrations between 4 and 15 μg/ml). The resistance of many members of the Halobacteriaceae to high concentrations of quinolone compounds may in part be due to the high magnesium concentrations present in the growth media. Haloferax volcanii was sensitive to 40 μg/ml ciprofloxacin when grown at suboptimal magnesium concentrations (0.1 M), but was hardly affected by 100 μg/ml of the inhibitor when grown in the presence of 0.5–0.75 M MgCl₂. It is suggested that the putative archaeal type II DNA topoisomerase has properties similar to those of the enzyme from Bacteria, although its sensitivity to quinolone antimicrobial compounds may be lower. Received: 6 November 1995 / Accepted: 26 February 1996     

Effects of<Emphasis Type="Italic">Fomitopsis pinicola</Emphasis> extracts on antioxidant and antitumor activities

We investigated the effects ofFomitopsis pinicola extract on biological activity by examining the antioxidant and antitumor activityin vitro andin vivo. When theF. pinicola extract concentration was raised from 60 to 120 μg/mL, the DPPH scavenging rate increased from 50.3 to 88.2% and the superoxide anion radical scavenging rate increased from 45.2 to 85.3% when theF. pinicola extract concentration was raised from 500 to 700 μg/mL. After incubatingF. pinicola extract for 12 h, the linoleic acid scavenging rate increased from 35.5 to 90.5%. A similar finding was observed for butylated hydroxytoluene. The total phenolic content of theF. pinicola extracts were approximately 10- to 16-fold higher than what was observed in theP. nebrodensis andA. camphorate extracts. The glutathione production, using decoctions prepared fromF. pinicola, was 20.0 μM/g of liver, which corresponded to approximately 4.0-fold higher than the control. The glutathione peroxidase activity was 8.3 U/mg of protein, which was approximately 2.8-fold higher than the activity level observed in the control rat livers. The cell viability rates of all the human cancer cells, when 100 μg/mL of ethanol extract was used for the different types of cancer cells, decreased with increasing extract concentrations in comparison to the hot water extract. In particular, when HeLa and Hep3B cells were incubated with 1.000 μg/mL of methanol extract, the cell viability rates were 20 and 25%, respectively, which was approximately 3.0-fold higher than what was observed for the hot water extract. The first two authors contributed equally to this work.     

In vitro inhibition of murine hematopoietic progenitors and stromal cells by vinorelbine

Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine bone marrow. The in vitro growth of colony-forming units–granulocyte/macrophage (CFU-GM), burst forming units–erythroid (BFU-E) and colony-forming units–mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 μg/ml) suppressed all of the different progenitor cells by 100%. A comparison of the dose–response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing doses of VRB. The appearence of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity was seen in cultures exposed to 0.05 and 0.075 μg/ml; and a marked loss at the dose of 0.1 μg/ml. Our results show that VRB has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected at a similar dose (0.025 μg/ml), suggesting that the stroma is more resistant to this drug. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen

Eighteen colorectal carcinoma patients without macroscopic disease after surgery were immunized using recombinant (r) human (h) carcinoembryonic antigen (CEA) with (n = 9) or without (n = 9) the addition of soluble granulocyte/macrophage-colony-stimulating factor (GM-CSF). The dose of rhCEA per immunization was 100 μg (n = 6), 316 μg (n = 6) or 1000 μg (n = 6). rhCEA was given s.c. on day 1 and 80 μg/day of GM-CSF s.c. on days 1–4. The schedule was repeated six times during a period of 9 months. All patients in the GM-CSF group developed a strong rhCEA-dose-dependent IgG antibody response while only one-third of the non-GM-CSF patients mounted a weak antibody response. All patients (9/9) in the GM-CSF group developed a strong rhCEA-specific proliferative T cell response as well as type I T cells (interferon γ secretion). In 45% of the patients also a weak type II T cell response (interleukin-4 secretion) was evoked. Both MHC-class-I- and -II restricted rhCEA-specific T cells were noted. A specific cellular response (proliferation and/or cytokine secretion) against native hCEA could be found in 8/9 patients in the GM-CSF group, although at a significantly lower level than against rhCEA. In the non-GM-CSF group a weak rhCEA-specific T cell response was induced. Three patients had a proliferative response, 4 patients type I T cells and 6 patients type II T cells. No signs of autoimmune reactions were noted. Local pharmacological administration of GM-CSF seemed to be a prerequisite for the induction of a strong immunity against baculovirus-produced hCEA protein. However, the cellular response against native CEA was of a significantly lower magnitude. Received: 13 November 1997 / Accepted: 21 May 1998     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Change in apparent and true absorption and retention of dietary zinc with age in rats

Two groups of 16 rats each were fed the same diet with 12.9 ppm Zn. Nine days after each animal was injected with⁶⁵Zn for assessing fecal zinc of endogenous origin, zinc intake and excretion were determined for a six-day period at the age of about five (group I) and nine (II) weeks. At mean growth rates of 5.1 and 5.2 g/day, food consumption per gram of gain was 2.01 g in group I vs 2.86 g in II. Overall, zinc retention amounted to 21 vs 25 μg Zn/g of gain. Apparent absorption averaged 92 vs 74% of Zn intake (132 vs 189 μg/day), while true absorption averaged 98 vs 92%. It was concluded that endogenous fecal zinc excretion was limited to the indispensable loss (F _em) in group I (7 μg/day), while it exceeded this minimum loss in group II (33 μg/day). True retention, which reflected total zinc utilization (true absorption times metabolic efficiency), was derived from apparent absorption plusF _em (11 μg/day for group II according to the greater metabolic body size of the rats). It averaged 98% of Zn intake in group I vs 80% in group II. The mean metabolic efficiency was 100% vs 87%. The conclusion was that these marked differences between age groups in utilizing the dietary zinc reflected the efficient homeostatic adjustments in absorption and endogenous excretion of zinc to the respective zinc supply status.     

Growth suppression of human transformed cells by treatment with bark extracts from a medicinal plant, Terminalia arjuna

Summary We have investigated the effects of acetone and methanol extracts of a medicinal plant, Terminalia arjuna, on the growth of human normal fibroblasts (WI-38), osteosarcoma (U2OS), and glioblastoma (U251) cells in vitro. We found that both extracts at 30 μg and 60 μg/ml concentrations inhibit the growth of transformed cells; the growth of normal cells was least affected. Although the transformed cells appeared to have fragmented nucleus by Hoechst staining, no deoxyribonucleic acid laddering effect was observed. In response to the extract treatment, the tumor suppressor protein, p53, was induced in U2OS but not in U251 and WI-38 cells. A cyclin-dependent kinase inhibitor, p21^WAF1, was induced in transformed cells only. The study suggests that the bark extract of medicinal plant, T. arjuna, has components that can induce growth arrest of transformed cells by p53-dependent and-independent pathways.     

Reduction of Hexavalent Chromium by Cell-Free Extract of Bacillus sphaericus AND 303 Isolated from Serpentine Soil

Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S₁₂, S₃₂, and S₁₅₀). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S₁₅₀) concentration equivalent to 4.56 mg protein/mL, 300 μM Cr(VI) and pH 6.0 after 30 min incubation at 30°C. The enzyme was heat labile; 80% of its activity was lost when exposed at 70°C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a V_max of 1.432 μmol Cr(VI)/mg protein/min and K_m of 158.12 μM Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity over control was recorded with 1.0 μM NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 μM Co(II), which retained 93% activity over control.