In vitro culture, transformation and genetic fidelity of Milk Thistle.

This review article presents a consolidated explanation and provides a comprehensive description of various studies, carried out on in vitro culture and hairy root cultures of S. marianum which can be consider an alternative source of flavonolignans. To overcome the constrains of conventional propagation of silybum plant, tissue culture and advanced biotechnology proved to be an influential tool that can complement conventional breeding and accelerate silybum development. The present review is focused on biotechnological tools like in vitro culture, hairy root cultures and genetic fidelity of S. marianum which can be a potent tool for production of secondary metabolites from these cultures.

Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization.

Different techniques were adopted for molecular characterization of several indigenous strains of Bacillus thuringiensis (Bt) previously isolated from Egyptian soil samples. These isolates show different toxicity levels against neonate larvae of both insect species; Spodoptera littoralis (Biosduval); and Helicoverpa armigera (Hübner). The parasporal crystals among the most potent isolates contained polypeptides of about 127 and 130 kDa. PCR screening for genes encoding different Cry genes was performed. The Cry 1 gene is the most abundant in these isolates (83.33%) among tested Cry-type genes, followed by Cry 1 gene subfamilies (Cry 1B and Cry 1C) with percentage of 38.88% and 77.77%, respectively. The tested isolates showed the presence of Cry 2A(a,b) gene, but not all of these isolates were positive for Cry 2 gene (55.55%). Only 27.77% and 16.66% of the tested isolates harbor Cry 4 and Cry 3 genes, respectively. All strains were negative in PCR assays for the Vip 3Aa1 gene. Moreover, DNA fingerprinting using RAPD-PCR was performed to detect the genetic similarities and dissimilarities among the different isolates and standard strains. Assessment of Bt diversity based on the combined analysis of their protein and RAPD-PCR banding patterns was performed. This study demonstrates that Bt strains isolated from Egyptian soil samples can be distinguished and identified on the basis of the distribution of Cry-type genes and RAPD fingerprints.

Histopathology of the larval midgut of Helicoverpa armigera (Hübner) fed on Bacillus thuringiensis crystals and Bt-tomato plants.

The histopathological effects of the spore-crystal complex of indigenous Bacillus thuringiensis (Bt) isolate, as well as Cry 2Ab gene expressed in transgenic tomato plants on the midgut of 4th instar larva of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidea) has been investigated using the transmission electron microscope (TEM). Remarkable ultrastructural changes were observed in the columnar and goblet cells of the larval midgut after feeding on either transgenic tomato leaves, or spore-crystal complex of Bt. The effects observed included breakdown of microvilli of epithelial cells, increase in the electron density of the cytoplasm and vacuolation associated with different sizes of lysosomes; interruption of the goblet cells and distorted goblet cavities which lost their cytoplasmic projections; destruction of the mitochondria which lost their cristae; degeneration of the endoplasmic reticulum; collapse of the nucleus associated with rupture of nuclear envelope and clumped chromatin. Feeding the larvae on transgenic Bt-tomato plants caused in addition to the aforementioned changes severe vacuolation and degeneration of the nucleus in both columnar and goblet cells and the nuclear membrane was broken into electron dense ring spheres.

Serum lipoprotein composition, lecithin cholesterol acyltransferase and tissue lipase activities in pregnant diabetic rats and their offspring receiving enriched n-3 PUFA diet.

The effects of dietary n-3 polyunsaturated fatty acids on lipoprotein concentrations and on lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL) and lecithin cholesterol acyltransferase (LCAT) activities were studied in streptozotocin-induced diabetic rats during pregnancy and in their macrosomic offspring from birth to adulthood. Pregnant diabetic and control rats were fed Isio-4 diet (vegetable oil) or EPAX diet (concentrated marine omega-3 EPA/DHA oil), the same diets were consumed by pups at weaning. Compared with control rats, diabetic rats showed, during pregnancy, a significant elevation in very low density lipoprotein (VLDL) and low and high density lipoprotein (LDL-HDL(1))-triglyceride, cholesterol and apoprotein B100 concentrations and a reduction in apoprotein A-I levels. HTGL activity was high while LPL and LCAT activities were low in these rats. The macrosomic pups of Isio-4-fed diabetic rats showed a significant enhancement in triglyceride and cholesterol levels at birth and during adulthood with a concomitant increase in lipase and LCAT activities. EPAX diet induces a significant diminution of VLDL and LDL-HDL(1) in mothers and in their macrosomic pups, accompanied by an increase in cholesterol and apoprotein A-I levels in HDL(2-3) fraction. It also restores LPL, HTGL and LCAT activities to normal range. EPAX diet ameliorates considerably lipoprotein disorders in diabetic mothers and in their macrosomic offspring.

Phototactic behavior in Daphnia magna Straus as an indicator of toxicants in the aquatic environment.

In this study, the phototactic behavior of Daphnia magna was investigated as a possible bioindicator for the following 11 chemicals commonly found in the aquatic environment: benzo(b)fluoranthene, mercury (II) chloride, dimethoate, lindane, linuron, MCPA, TBTO, carbon tetrachloride, thiram, 2,4,6-trichlorophenol and arsenic trioxide. Phototactic response was monitored as the movement of 7 to 8 day-old D. magna individuals. The analysis was carried out using glass test tubes divided radially into two zones, with increasing distance from a light source. For each of the compounds, different concentrations and exposure times were analyzed, and the behavior of the D. magna in each of the treatments compared to the controls in which the chemicals were not added. Using the experimental model described here, all of the 11 chemicals could be detected following exposure times of between 15 min and 48 h. The lowest concentrations detected using this technique were between 2 and 43 times lower than the LC(50) and EC(50) values reported for D. magna. The results of this study show that the analysis of phototactism is a useful method for detecting the presence of a wide range of potentially toxic chemicals found in the aquatic environment.

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river.

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.

Detection of microcystin synthetase genes in health food supplements containing the freshwater cyanobacterium Aphanizomenon flos-aquae.

In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.1--4.72 microgg(-1) (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria.

Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction.

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 micro g l(-1).

Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins.

The Artemia salina bioassay was successfully applied to the analysis of the hepatotoxic cyanobacterial alkaloid and protein synthesis inhibitor, cylindrospermopsin. A dose-dependent response in mortality was observed for purified cylindrospermopsin and LC(50) values decreased with time from 8.1 to 0.71 microg/ml(-1), between 24 and 72 h, respectively. Cylindrospermopsin was slightly less potent than micro cystin-LR, with similar LC(50) values on a gravimetric basis, but was more toxic to A.salina than the protein synthesis inhibitors, cycloheximide, chloramphenicol and tetracycline. Cylindrospermopsin-containing strains of the cyanobacterium Cylindrospermopsis raciborskii were found to be toxic to A.salina and the LC(50) concentration for these strains over time was greater than the LC(50) for purified cylindrospermopsin, with the exception of C. raciborskii strain CR1.

The effect of low-dose combined oral contraceptive pills versus injectable contraceptive (Depot Provera) on liver function tests of women with compensated bilharzial liver fibrosis.

This was an open comparative trial of two types of hormonal contraceptives, a low-dose combined oral contraceptive and the injectable depot-medroxyprogerone acetate, with a control group using an intrauterine device (copper T-380). Clients were recruited from bilharzial patients attending the maternal-child health and family centers affiliated with the National Liver Institute. Clients with compensated bilharzial liver disease were chosen and were divided into three subgroups according to their own preference for the method of contraception. Baseline, 2-, and 4-months liver function tests were compared, and abdominal ultrasound scanning of the liver was done. There was a nonsignificant change in liver function tests, and the portal vein diameter measured by ultrasound scanning was not changed with steroidal contraceptives. The use of combined oral contraceptive containing low-dose estrogen or using depot medroxyprogertone acetate injectables are safe and can be prescribed in cases with compensated bilharzial hepatic fibrosis with normal function.

Failed primary management of iatrogenic biliary injury: incidence and significance of concomitant hepatic arterial disruption.

BACKGROUND: Vasculobiliary injury (VBI) is a well-recognized complication of laparoscopic cholecystectomy (LC). In patients with failed primary management of bile duct injury (BDI), an assessment of the hepatic arterial system may be important to determine the presence of VBI. This study was conducted to determine the incidence of VBI in patients with failed primary management of LC-related BDI and to establish a potential correlation between the level of BDI and the incidence of VBI. METHODS: A retrospective review was conducted on 18 patients referred for failed primary management of LC-related BDI who underwent prospective arteriography as part of the preoperative work-up. RESULTS: Of the 18 patients who sustained BDI, Bismuth level 4 lesions were found in 7 patients (39%), level 3 in 8 patients (44%), and level 2 in 3 patients (17%). VBI was identified on arteriography in 11 patients (61%). VBI was present in 71% of patients with level 4 lesions, 63% of patients with level 3 lesions, and 33% of patients with level 2 lesions. The time interval from primary management to its failure was longer in VBI than in BDI alone. CONCLUSIONS: We have observed a high incidence of VBI in patients with failed primary management of LC-related BDI. Arterial disruption may affect the outcome of primary management of BDI.

Three-dimensional MR pulmonary perfusion imaging and angiography with an injection of a new blood pool contrast agent B-22956/1.

Initial evaluation of a new blood pool agent, B-22956/1, for pulmonary imaging was performed in five domestic pigs with artificial embolism. Pre-embolism 3D pulmonary perfusion images were first acquired by injecting an extravascular agent, gadoteridol. The pulmonary arteries of the pigs were then occluded by the artificial emboli. Post-embolism perfusion scans were subsequently performed by injecting B-22956/1. Additional post-embolism high-spatial-resolution angiograms were also acquired. Parenchyma perfusion deficits were well depicted in the post-embolism perfusion maps. The post-embolism angiography clearly revealed the location and extent of the filling defects in the pulmonary vessels. Signal intensities of perfusion maps on the normal parenchyma were significantly improved (30%) by using B-22956/1, in comparison with perfusion images using gadoteridol (P < 0.01). Many pulmonary angiograms with approximately equal contrast could be obtained even at 22 minutes after the injection of B-22956/1. Our initial results indicate that blood pool agent B-22956/1 may provide opportunities for whole-lung-coverage perfusion mapping and additional high-resolution target angiograms after a single injection.

Effect of MR angiography on the diagnosis and treatment of patients with suspected renovascular disease.

PURPOSE: Although the diagnostic accuracy of renal magnetic resonance (MR) angiography is established, its effect on referring physicians is unknown. The authors prospectively measured the effect of MR angiography results on referring physicians' diagnosis and treatment (plans) of patients with suspected renovascular disease. MATERIALS AND METHODS: Referring physicians prospectively completed questionnaires before and after MR angiography was performed during evaluation of their patients with suspected renovascular disease. The questionnaires asked them to estimate the probability (0%-100%) of their most likely diagnosis before and after receiving the imaging information. They were also asked for their anticipated and final treatment plans. The authors calculated the mean gain in diagnostic percentage confidence and the proportion of patients with changed initial diagnoses or anticipated management. A paired t-test was used to assess significance of the gains in diagnostic percentage confidence. RESULTS: Physicians prospectively completed pre- and post-MR-angiography questionnaires for 30 patients. MR angiography improved mean diagnostic certainty by 35% (P < .0001). MR angiography changed physicians' initial diagnoses in 12 patients (40%). Anticipated treatment plans were changed in 20 patients (67%). Invasive procedures were avoided in eight patients (27%). CONCLUSION: MR angiography has a substantial effect on the diagnostic and therapeutic decision-making of physicians managing patients with suspected renovascular disease.

The vascular impact of uterine artery embolization: prospective sonographic assessment of ovarian arterial circulation.

PURPOSE: The etiology of premature ovarian failure after uterine artery embolization (UAE) is unknown. The authors prospectively assessed ovarian arterial circulation before and after UAE with use of ultrasonography (US). The authors hypothesize that nontarget embolization of the ovary occurs during routine UAE. MATERIALS AND METHODS: Twenty-three women (mean age of 42.6 years; range, 35-51 y) participated in the study. Grayscale, color, and pulsed-wave Doppler US studies were performed immediately before and after UAE. Resistive index (RI) and pulsatility index (PI) were calculated. The proportion of women who developed increased vascular impedance after UAE was statistically assessed with use of the Yates-corrected chi(2) test. RESULTS: Seventeen of 23 patients (74%) completed the study. Nine of 17 (54%) showed complete loss of ovarian arterial perfusion after UAE. Six of 17 (35%) had increases in RI and PI, whereas two of 17 (11%) had decreases in RI and PI. The increase in vascular impedance after UAE in 15 of 17 patients was significant (P <.0001). CONCLUSION: Loss of detectable ovarian arterial perfusion occurs in the majority of patients undergoing UAE. Ovarian vascular impedance increases in nearly all patients as a result of UAE. The authors conclude that inadvertent nontarget embolization of the ovarian arterial bed occurs during routine UAE.

Varied diazotrophies, morphologies, and toxicities of genetically similar isolates of Cylindrospermopsis raciborskii (nostocales, cyanophyceae) from Northern Australia.

The potentially toxic freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO3-, NH4+, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven C. raciborskii isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH4+ as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH4+. Cultures supplied with NO3- were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of C. raciborskii trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH4+ and the statistically significant increase in vegetative cell length (nitrogen depleted < NO3- < NH4+). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications for Cylindrospermopsis taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of C. raciborskii.

Treatment of stomal variceal hemorrhage with TIPS: case report and review of the literature.

We report a case in which recurrent hemorrhage from stomal varices was successfully treated by placement of a TIPS in a patient with prior colectomy for inflammatory bowel disease. Although several treatment options have been reported for this entity we believe that TIPS offers minimally invasive and definitive treatment.

The impact of uterine fibroid embolization on resumption of menses and ovarian function.

PURPOSE: To evaluate the effect of uterine fibroid embolization (UFE) on menstruation and ovarian function. METHODS: The authors performed an observational study of UFE for the treatment of symptomatic fibroids. All patients had regular predictable menses before intervention and none had clinical or laboratory findings of menopause. UFE was performed with use of standard methods with 355-700-microm-diameter polyvinyl alcohol (PVA) foam particles. The incidence of ovarian failure was calculated for women younger than 45 years and for those 45 years or older, based on retrospective stratification by age. The authors assessed statistical differences in ovarian failure between the two age groups with use of the X2 test. RESULTS: Sixty-six premenopausal women (age range, 30-55 years) underwent bilateral UFE and were followed for an average of 21 weeks (range, 12-77 weeks). In 56 of 66 (85%) patients, regular menses resumed after an average of 3.5 (range, 1-8) weeks. In 10 of 66 (15%) patients, regular menses did not resume. Clinical and biochemical findings consistent with ovarian failure and presumed menopause were seen in nine of 10 patients without resumption of menses (14% of total patients). Ovarian failure occurred in nine of 21 (43%) women older than 45 years and in none of the 45 women younger than 45 years (P < .05). There were no differences in presenting symptoms, amount of PVA used, or fibroid size between patients who did and did not resume menses. CONCLUSION: The majority of patients undergoing UFE will have resumption of menses, but the incidence of postprocedure ovarian failure is considerably higher than reported to date. Loss of menses induced by UFE is significantly more likely to occur in women older than 45 years.

The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus.

Redclaw crayfish, Cherax quadricarinatus harvested from an aquaculture pond infested by a bloom of the cyanobacterium Cylindrospermopsis raciborskii (order: Nostocales), were shown to accumulate the toxic alkaloid cylindrospermopsin. Pond water samples collected during the bloom contained 589 microg l(-1) of the toxin (93% in the cyanobacterial cells, 7% in the water). Crayfish from the pond contained cylindrospermopsin at concentrations of 4.3 microg g freeze dried hepatopancreas tissue and 0.9 microg g freeze dried muscle tissue. Trichomes of C. raciborskii were observed in gut contents of crayfish harvested during the cyanobacterial bloom, indicating that the most likely mechanism for accumulation of the toxin was by ingestion of cyanobacterial cells. Crayfish subjected to an extract of harvested bloom material under laboratory conditions for a period of 14 days were also found to accumulate cylindrospermopsin, indicating that this toxin is also absorbed into the tissues by direct uptake of the toxin in solution.