TheAspergillus parasiticus polyketide synthase genepksA,a homolog ofAspergillus nidulans wA,is required for aflatoxin B1 biosynthesis

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced byAspergillus parasiticus andAspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway inA. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipientA. parasiticus genome at a specific locus, designatedpksA. Sequence analysis suggest thatpksA is a homolog of theAspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conservedβ-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of thepksA product. Noβ-ketoacyl reductase and enoyl reductase domains were found, suggesting thatpksA does not encode catalytic activities for processingβ-carbon similar to those required for long chain fatty acid synthesis. ThepksA gene is located in the aflatoxin pathway gene cluster and is linked to thenor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose thatpksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

Summary An undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented with 10% fetal bovine serum (FBS). It is believed that certain, as yet unidentified, serum components play critical roles in the attachment and proliferation of MSCs. Commercially available FBS is poorly characterized and may vary in composition and quality from lot to lot. This study describes a method for the selection of lots of FBS that best support maintenance of the undifferentiated state, mitotic expansion of MSCsin vitro, and retention of multilineage developmental potential in response to appropriate cues.     

Effects of antifilarial drugs on the activities of the oxidative enzymes of mitochondria and microsomes of rat liver and kidneys

Centperazine or diethylcarbamazine, administered at various dose levels to rats inhibited the activity of succinate dehydrogenase significantly in 4 hrs in liver. Centperazine also inhibited the activity of cytochrome-c oxidase but stimulated the activity of benzo (a) pyrene hydroxylase in liver. In kidneys, activities of succinate dehydrogenase, cytochrome-c oxidase and aniline hydroxylase were significantly inhibited by centperazine only, however, the activity of benzo (a) pyrene hydroxylase was inhibited by both the drugs. These drugs had no effect on the activity of aminopyrene N-demethylase and cytochrome P-450 contents of liver and kidneys.     

Effect of dihydroergotoxine, a cerebral vasodilator, on cognitive deficits induced by prenatal undernutrition and environmental impoverishment in young rats

The study was conducted on 64 Charles Foster strain albino rats, which were equally distributed into 8 evenly matched groups, following a 2 x 2 x 2 factorial design, by varying three independent factors at two levels: nutrition--normal and undernutrition; environment--enrichment and impoverishment, and drug treatment--vehicle and dihydroergotoxine (3 mg/kg, i.p.). Prenatal undernutrition was induced by restricting the mother's food intake. The environmental enrichment/impoverishment and the vehicle/dihydroergotoxine treatments were given during the postweaning period of the pups. The rats were subjected to original and subsequent reversal brightness discrimination learning tests in a single unit T-maze at 8-9 weeks of age. Thereafter, the animals were tested for passive avoidance learning. The results indicate that undernutrition caused significant original and reversal discrimination learning, deficits whereas environmental deprivation attenuated only the original discrimination learning performance. Dihydroergotoxine treatment facilitated the learning performance of rats in both the original and reversal learning tests. Nutritional, environmental and dihydroergotoxine treatments had no effect on the retention of the passive avoidance learning, both at 24 hr and 1 week intervals. Dihydroergotoxine treatment attenuated the learning deficits induced by prenatal undernutrition. The results indicate that dihydroergotoxine is not likely to be useful in cognitive deficits, induced by malnutrition, though it facilitated learning acquisition, since it had no effect on retention.     

Xanthone and flavonol constituents of Swertia hookeri

The whole plant of Swertia hookeri, collected at flowering has been shown to contain two tri- and nine tetraoxygenated free, glucosyloxy, and stearyl ester xanthones and one flavonol stearyl ester. Among these, three are previously unreported in nature and one was known previously only as a synthetic compound. The xanthones are based on 1,3,5,-, 1,3,5,8- and 1,3,7,8-oxygenated systems with the middle oxygenation pattern predominating. The two ester compounds appeared only at the flowering stage. Plants collected at the pre-flowering stage gave the corresponding free compounds. The biochemical and biological significance of these findings are appraised.     

Effect of electrical stimulation of the olfactory tract and the caudal spinal cord on the Dahlgren cells in Clarias batrachus L.

Electrical stimuli applied to the olfactory tract for one minute caused partial depletion, but for two to five minutes resulted in complete depletion of the neurosecretory material (NSM) from the Dahlgren cells as well as from the urophysis. However, if similar stimuli were directly applied to the caudal spinal cord for one minute, the NSM was completely depleted. The neurosecretory granules were reaccumulated in the neurons within fifteen minutes after the stimuli were cut of A rapid depletion of the NSM from the caudal neurons was correlated with their electrical properties and rapid transduction of nervous information into the hormonal message. The immediate response of the caudal neurons to the olfactory tract stimulation suggested that the former are synaptically controlled by a center in the brain.     

Oncosecretomics coupled to bioenergetics identifies α-amino adipic acid, isoleucine and GABA as potential biomarkers of cancer: Differential expression of c-Myc, Oct1 and KLF4 coordinates metabolic changes

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.