Airborne cytotoxicity in the DiSC assay caused by solutions of treosulfan but not busulphan

Treosulfan and busulphan are similar molecules, the former used in the treatment of ovarian cancer and the latter in chronic myelogenous leukaemia. We have used both in the differential staining cytotoxicity (DiSC) assay forin vitro drug sensitivity testing to aid in the choice of chemotherapy for individual patients.It was observed that occasionally the viability of control cells in one assay box was reduced compared with control cells in other boxes from the same assay. Treosulfan was suspected as the cause because cells throughout the microtitre box containing treosulfan had reduced viability in 28/62 (45%) experiments and in 9 of these, total kill of all cells in the box was observed.We tested the hypothesis that a metabolite of treosulfan might be the cause of this airborne cytotoxicity, and found that whilst 10 mg ml^–1 of either methane sulphonic acid or tetrahydrofuran had no airborne cytotoxic effect, 1 mg ml^–1 diepoxybutane killed over 95% of cells in all tubes in the same box.Treosulfan is another chemical (cf. azide, mafosfamide and possibly other cytotoxic agents) that can cause airborne cytotoxicity.Abbreviations ALL acute lymphoblastic leukaemia - AML acute non-lymphocytic leukaemia - CLL chronic lymphocytic leukaemia - NHL non-Hodgkin's lymphoma - DiSC assay differential staining cytotoxicity assay - MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay - PBS phosphate buffered saline     

Synergistic antidigestion effect of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> and bovine colostrums in simulated gastrointestinal tract (in vitro)

Probiotics and bovine colostrums had been proven to be beneficial for human health. Lactobacillus rhamnosus ZDY114 and anti-Helicobacter pylori bovine colostrums were used for the preparation of microecological additives, and their synergistic antidigestion effect in the simulated gastrointestinal tract (in vitro) was investigated. Either L. rhamnosus or purified IgG from immune colostrums was very sensitive in simulated gastric environment and slightly sensitive in simulated intestinal tract. No viable counts were recovered from the solution of dissolved freeze-dried powder (7.14 log₁₀ CFU/ml) of L. rhamnosus when digested at pH 3.0 with pepsin for 30 min. Activity of purified IgGs from immune colostrums could not be detected when digested at pH 3.0 with pepsin for 30 min; 29% titer could be detected when digested at pH 8.0 with trypsin for 5 h. The IgGs in nonpurified immune colostrums presented stronger resistance against gastrointestinal digestion than purified IgGs. Moreover, the combination of L. rhamnosus ZDY114 and immune colostrums strengthened their antidigestion ability. Even under pH 3.0, 4.0 with pepsin, the titer of anti-HP IgG maintained above 123 and 83.3%, respectively. Similarly, that titer was above 93.3% when digested at pH 8.0 with trypsin for 4.5 h. In conclusion, L. rhamnosus and anti-Helicobacter pylori bovine colostrums had synergistic antidigestion effect in simulated gastrointestinal tract (in vitro).     

Escherichia coli K12 does not colonize the gastrointestinal tract of Fischer-344 rats

Summary The colonizing potential ofEscherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 g/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any rat from any group. Thus, theE. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.     

Co-polymeric hydrophilic nanospheres for drug delivery: release kinetics, and cellular uptake

Nanobiotechnology focuses on the biological effects and applications of nanoparticles that include nano-safety, drug encapsulation and nanotherapeutics. The present study focuses on hydrophilic nanospheres of copolymers N-isopropylacrylamide [NIPAAM] and vinyl pyrrolidone [VP], encapsulating a bioactive derivative of 5-fluorouracil-hexyl-carbamoyl fluorouracil (HCFU). The size of the nanospheres was approximately 58 nm and the surface charge measured was -15.4 mV. Under optimal conditions, the yield was >80%, and the drug loading was 2%. The entrapment efficiency was approximately 75%. Wide-angle X-ray diffraction analysis showed that the entrapped HCFU was present in an amorphous state, which has higher water solubility compared with the crystalline state. Slow drug release from nanospheres was observed in PBS and serum, with approximately 80% released at 37 degrees C after 72 h. The HCFU loaded polymeric nanospheres have been found to be stable in whole blood having negligible RBC toxicity. Cytotoxicity in Mia-Paca 3, pancreatic cancer cell line was done in a 24-72 h assay. Dose dependant cytotoxicity was observed when incubated with various concentrations of HCFU loaded polymeric nanospheres while HCFU per se (<1 mg) showed 90% toxicity within 24 h.     

Expression of pro-apoptotic Bfk isoforms reduces during malignant transformation in the human gastrointestinal tract

Reduced expression of pro-apoptotic Bcl-2 family proteins has been described in many gastrointestinal cancers, and may play a role in tumourigenesis. The human homologue of the pro-apoptotic Bcl-2 protein, Bfk, is predominantly expressed in tissues of the gastrointestinal tract. In colon, four alternatively spliced isoforms were identified; of which two are pro-apoptotic when overexpressed. In the transition from normal tissue to tumour, pro-apoptotic Bfk isoform expression is substantially reduced in up to 80% of tumours isolated from the human gastrointestinal tract (8/10 colonic tumours and 26/37 of all gastrointestinal tumours) compared to 3/117 tumours from outside the gastrointestinal tract. These data suggest that pro-apoptotic isoforms of Bfk may help to protect against the development of human gastrointestinal malignancy.     

Generation of cytotoxic lymphocytes in mixed lymphocyte reactions II. Importance of private and publicH-2 alloantigens on the expression of cytotoxicity

MLC were established to test for the generation of specific cytotoxic effector cells in CML. The target cell used to assay for CML in the five combinations tested was of a differentH-2 haplotype from the stimulating cell population. Cytotoxicity was observed against this target only when it shared private alloantigens (antigens that are specific for theH-2D andH-2K region of differentH-2 haplotypes) with the stimulating cell population. Very weak or no Cytotoxicity was found when such alloantigens were not shared, although cross-reactive publicH-2 specificities were. These findings indicate that T cells display a cytotoxic potential against privateH-2 antigens in a primary response in vitro and are not capable of responding to publicH-2 specificities to the same level.BSS balanced salt solution - CML cell-mediated lympholysis - GPC guinea pig complement - ¹²⁵IUdR ¹²⁵I-iodo-deoxyuridine - MLC mixed lymphocyte culture - SE standard error     

Growth inhibition and induction of differentiation and apoptosis mediated by sodium butyrate in caco-2 cells with algal glycolipids

Summary Glycolipids should have potential effects as antitumor agents. However, very few studies have examined this property of digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) on colon cancer cells. Cell viability was determined every 24 h with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay up to 72 h. Alkaline phosphatase activity was measured for assessing cell differentiation. Apoptosis was tested with enzyme-linked immunosorbent assay analysis. Growth of Caco-2 cells was inhibited apparently at 48 h after addition of SQDG and at 72 h with DGDG. Alkaline phosphatase activity of Caco-2 cells obviously increased in combination with DGDG or SQDG and sodium butyrate (NaBT) at 72 h, indicating that DGDG and SQDG enhanced cell differentiation induced with NaBT. An increased enrichment factor was found when the cell was treated in combination with DGDG or SQDG and NaBT. These results strongly suggest that DGDG and SQDG should be considered as the leading compounds of potentially useful colon cancer chemotherapy agents when NaBT is combined.     

An efficient protocol for transient transformation of intact fruit and transgene expression inCitrus

In this study, conditions were optimized for transient gene expression in Rough Lemon (Citrus jambhiri Lush.), a major rootstock used in the citrus growing regions of Pakistan.Agrobacterium tumefaciens carrying the binary vector p35GUSINT, containingNPT II andGUS genes, was used in the study. The transformation method was based on injection ofAgrobacterium intoCitrus fruits followed by histochemical assay ofGUS activity in different tissues. Different tissues of mature fruits exhibited significantly different percentages of transientGUS expression: in rind (76%), spongy tissue (92%), juice vesicles (0%) and seeds (83%) (P<0.01)., The incubation period after injecting theAgrobacterium culture also showed a significant (P<0.01) effect on the transient expression ofGUS in these tissues. An incubation period of 48 h was found to be the best (72%) for transformation of whole fruit, followed by 72 h (67%) and 96 h (49%). TransientGUS expression also varied significantly (P<0.01) in juice vesicles and seeds as fruit matured. Juice vesicles from mature fruits showed no transientGUS expression, while those from immature fruits showed 50% expression. Furthermore, transformation of seeds had no effect on their germination capability. Germinating seeds from mature fruits injected withAgrobacterium culture showed tolerance to kanamycin (100 mg/L), which varied with the incubation period (55% at 48 h, 25% at 72 h and 23% at 96 h). This report offers an easy protocol for transient expression studies of transgenes and has the potential to be used for stable transformation ofCitrus.     

Relationships in Canada between mortality from tumours of the gastrointestinal tract and marital status.

The mortality from tumours of the gastrointestinal tract in the Canadian population in 1970-72 was 16% higher in single than in married men (on the basis of age-adjusted rates), 25% higher in widowed men and 28% higher in divorced men. All these differences were unlikely to be due to chance. The rates were 4% higher for single women, 14% higher for widows and 22% higher for divorced women, compared with the married. The differences for single and divorced women were not significant. Substantial excess mortality was found in the unmarried for tumours of the mouth, pharynx and esophagus, and rectum; for tumours of the stomach and colon the excess was small or nonexistent. This variation between sites suggests that systematic errors in the census data used as denominators are not responsible for the high mortality for the unmarried from certain tumours. The effect is found in conditions for which treatment can have made little difference (e.g., a 75% excess mortality for tumours of the esophagus in single men compared with married) and in conditions for which differences in the use of medical facilities may have been important (e.g., a 44% excess mortality for tumours of the rectum in widowers).     

Gastrointestinal assimilation of Cu during digestion of a single meal in the freshwater rainbow trout (Oncorhynchus mykiss)

Gastrointestinal processing and assimilation of Cu in vivo was investigated by sequential chyme analysis over a 72 h period following ingestion of a single satiation meal (3% body weight) of commercial trout food (Cu content=0.42 micromol g(-1)) by adult rainbow trout. Leaded glass ballotini beads incorporated into the food and detected by X-ray radiography were employed as an inert marker in order to quantify net Cu absorption or secretion in various parts of the tract. Cu concentrations in the supernatant (fluid phase) fell from about 0.06 micromol mL(-1) (63 microM) in the stomach at 2 h to about 0.003 micromol mL(-1) (3 microM) in the posterior intestine at 72 h. Cu concentrations in the solid phase were 10 to 30-fold higher than in the fluid phase, and increased about 4-fold from the stomach at 2 h to the posterior intestine at 72 h. By reference to the inert marker, overall net Cu absorption from the ingested food by 72 h was about 50%. The mid-intestine, and posterior intestine emerged as important sites of net Cu and water absorption and a potential role for the stomach in this process was also indicated. The anterior intestine was a site of large net Cu addition to the chyme, probably due to large net additions of Cu-containing fluids in the form of bile and other secretions in this segment. The results provide valuable information about sites of Cu absorption and realistic concentrations of Cu in chyme fluid for future in vitro mechanistic studies on Cu transport in the trout gastrointestinal tract.     

Barley-based medium for the cost-effective production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Summary Studies conducted for the multiplication of Bacillus thuringiensis (Bt) using barley Hordeum vulgare as the carbon source led to the development of a protocol for the cost-effective, mass production of Bt. The production employs the simple shake flask method and can be easily adopted with a production potential of 1.5 kg Bt per day approximately at an overall production cost of Rs. 360 kg^-1 (8 US dollars). The protocol is suitable for promoting localized production of Bt at the village/district level. The product when tested as 0.1% (w/v) spray against the castor semilooper, Achoea janata proved highly effective, causing immediate feeding cessation of the larvae followed by 85% and 100% mortality by 48 and 72 h after treatment, respectively.     

Human cell lines as models for multidrug resistance in solid tumours

In spite of our expanding knowledge on the molecular biology of cancer, relatively little progress has been made in improving therapy for the solid tumours which are major killers, e.g., lung, colon, breast. Significant advances over the past 10–15 years in chemotherapy of some tumours such as testicular cancer and some leukaemias indicates that, in spite of the undesirable side-effects, chemotherapy has the potential to effect cure in the majority of patients with certain types of cancer. Multidrug resistance, inherent or acquired, is one important limiting factor in extending this success to most solid tumours.In vitro studies described in this review are now uncovering a diversity of possible mechanisms of cross-resistance to different types of drug. Sensitive methods such as immunocytochemistry, RT-PCR orin situ RNA hybridisation may be necessary to identify corresponding changes in clinical material. Only by classifying individual tumours according to their specific resistance mechanisms will it be possible to define the multidrug resistance problem properly. Such rigorous definition is a prerequisite to design (and choice on an individual basis) of specific therapies suited to individual patients. Since a much larger proportion of cancer biopsies should be susceptible to accurate analysis by the immunochemical and molecular biological techniques described above than to direct assessment of drug response, it seems reasonable to hope that this approach will succeed in improving results for cancer chemotherapy of solid tumours where other approaches such as individualisedin vitro chemosensitivity testing have essentially failed. Results from clinical trials using cyclosporin A or verapamil are encouraging, but these agents are far from ideal, and reverse resistance in only a subset of resistant tumours. Proper definition of the other mechanisms of MDR, and how to antagonize them, is an urgent research priority.Abbreviations MDR multiple drug resistance - P-170=pgp P-glycoprotein=product ofmdr-1 gene     

Impact of metal oxide nanoparticles on oral release properties of pH-sensitive hydrogel nanocomposites

In this article, modified κ-carrageenan hydrogel nanocomposites were synthesized to increase the release ability of carrageenan hydrogels under gastrointestinal conditions. The effect of MgO nanoparticle loading in a model drug (methylene blue) release is investigated. Characterization of hydrogels were carried out using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Field Emission Scanning Electron Microscope (FESEM) and Differential Scanning Calorimetry (DSC). Genipin was used to increase the delivery performance in gastrointestinal tract delivery by decreasing release in simulated stomach conditions and increasing release in simulated intestine conditions. It is shown that the amount of methylene blue released from genipin-cross-linked nanocomposites can be 67.5% higher in intestine medium and 56% lower in the stomach compared to κ-carrageenan hydrogel. It was found that by changing the nanoparticle loading and genipin concentration in the composite, the amount of drug released can be monitored. Therefore, applying nanoparticles appears to be a potential strategy to develop controlled drug delivery especially in gastrointestinal tract studies.     

Chromosomal and plasmid encoded drug resistances of a <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> UTI 2 strain isolated from urine of a post-operative patient

The multi drug resistance Klebsiella pneumoniae in urinary tract infection is a common clinical problem in developing country like India. Use of random antibiotics, resulting multi drug resistance development, creates difficulties for treatment. In our present study, we investigated a strain of Klebsiella pneumoniae UTI 2 with multiple drug resistance, which was isolated from urine of a post operative woman patient (50 years) suffering from urinary tract infection with high fever. This strain is resistant to 36 antibiotics and sensitive to cefotaxime (Ce) and imipenem (I). After curing of plasmids, we observed that, 55% of drug resistant loci of K. pneumoniae UTI 2 are chromosomal and 40% are plasmid encoded. The organism is sensitive to 5% of drugs tested, i.e. Ce and I. This study contributes to understand the drug resistance of Klebsiella pneumoniae, which will enable better clinical management of catheter-associated urinary tract infections, a major health problem.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.     

Bacterial translocation from the gastrointestinal tracts of mice receiving immunosuppressive chemotherapeutic agents

Specific pathogen-free (SPF) mice were treated with certain classes of immunosuppressive chemotherapeutic agents to determine if they would promote bacterial translocation from the gastrointestinal tract to the mesenteric lymph node, spleen, or liver. The antimetabolites methotrexate, 5-fluorouracil, and cytosine arabinoside were injected once intraperitoneally into SPF mice, and the mice were tested for bacterial translocation from the gastrointestinal tract. When total organs from the treated mice were compared with the total organs from the control mice, the alkylating agent cyclophosphamide promoted bacterial translocation when injected once intraperitoneally at doses of 100–400 mg/kg. Increasing the number of injections of cyclophosphamide did not increase the incidence of bacterial translocation. The steroid prednisone also promoted bacterial translocation after one intraperitoneal injection of 10–150 mg/kg. Prednisone and cyclophosphamide at various doses appeared to be more effective in promoting bacterial translocation from the gastrointestinal tract than the antimetabolites. The aerobic and facultatively anaerobic bacteria translocating to the various organs were identified asLactobacillus acidophilus, Escherichia coli, Klebsiella pneumoniae, Streptococcus faecalis, Staphylococcus aureus, andProteus mirabilis. Groups of SPF mice also were injected once intraperitoneally with the minimal dose of each chemotherapeutic drug that induced bacterial translocation, and then tested for immune responsiveness toE. coli vaccination. Each of the chemotherapeutic agents at the minimal doses promoting bacterial translocation also suppressed the serum antibody responses to antigens of indigenousE. coli. However, other toxic manifestations of these chemotherapeutic agents also may be involved in promoting bacterial translocation. The promotion of bacterial translocation from the gastrointestinal tract by these chemotherapeutic agents has important implications for the pathogenesis of infectious disease in patients receiving these drugs.     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

Induction and decay of thermosensitivity in the flesh fly,Sarcophaga crassipalpis

When pharate adults of the flesh fly Sarcophaga crassipalpis are exposed to 40°C for 4 h they become more tolerant of high temperatures that are normally lethal (thermotolerance). In contrast, a 1-h exposure to 45°C decreases tolerance to a subsequent high temperature challenge (thermosensitivity). While control flies experience little mortality when held at 35°C for 24–48 h the thermosensitized flies die when exposed to 35°C. Sensitivity to a second thermal challenge slowly decays over a 72-h period. The acquisition of thermotolerance prevents the development of thermosensitivity. Brains from thermosensitized flies cultured at 43°C express the 72-kDa heat-shock protein and normal protein synthesis is inhibited. This implies that development of thermosensitivity is not associated with a loss in the capacity to express the 72-kDa heat-shock protein.Abbreviations ICN ICN Biomedicals, Inc. PO Box 19536, Irvine, CA 92713-9921 - LD light dark cycle - LT₅₀ time required to kill 50% of the test animals - SDS sodium dodecyl sulfate - TRIS Tris(hydroxymethyl)aminomethane     

Cytoprotective Effect of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> Extract on an In Vitro Experimental Model of Parkinson Disease

Parkinson′s disease (PD) is one of the most important neurodegenerative worldwide disorders. The potential cytoprotective effects of aqueous extract of Valeriana officinalis on rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells were demonstrated. The cytotoxicity, cell viability and analysis of cellular morphology were performed by MTT-tetrazole (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and phase contrast microscopy, respectively. Significant changes in the cellular morphology, and condensation of the cell body could be observed when cells were treated with 300 nM rotenone for 48 h. Three different concentrations of Valeriana officinalis extract were used (0.049, 0.098 and 0.195 mg/mL). These extracts brought about an increase of 7.0 ± 1.3%, 14.5 ± 1.3% and 14.5 ± 3.2% in cell viability. Our results indicated that neuroprotector action of the Valeriana officinalis extract provides support for later studies as they help understanding this drug for the development of cytoprotective various therapies in PD.