Evaluation of spore inoculum and confirmation of pathway genetic blueprint of T13αH and DBAT from a Taxol-producing endophytic fungus.

Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L-1 and 128.3 µg L-1, respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and baccatin III.

Identification and Detection of Bioactive Peptides in Milk and Dairy Products: Remarks about Agro-Foods.

Food-based components represent major sources of functional bioactive compounds. Milk is a rich source of multiple bioactive peptides that not only help to fulfill consumers 'nutritional requirements but also play a significant role in preventing several health disorders. Understanding the chemical composition of milk and its products is critical for producing consistent and high-quality dairy products and functional dairy ingredients. Over the last two decades, peptides have gained significant attention by scientific evidence for its beneficial health impacts besides their established nutrient value. Increasing awareness of essential milk proteins has facilitated the development of novel milk protein products that are progressively required for nutritional benefits. The need to better understand the beneficial effects of milk-protein derived peptides has, therefore, led to the development of analytical approaches for the isolation, separation and identification of bioactive peptides in complex dairy products. Continuous emphasis is on the biological function and nutritional characteristics of milk constituents using several powerful techniques, namely omics, model cell lines, gut microbiome analysis and imaging techniques. This review briefly describes the state-of-the-art approach of peptidomics and lipidomics profiling approaches for the identification and detection of milk-derived bioactive peptides while taking into account recent progress in their analysis and emphasizing the difficulty of analysis of these functional and endogenous peptides.

Fungal 7-epi-10-deacetyltaxol produced by an endophytic Pestalotiopsis microspora induces apoptosis in human hepatocellular carcinoma cell line (HepG2).

BACKGROUND: Paclitaxel (taxol) is a potent anticancer drug that is used in the treatment of a wide variety of cancerous. In the present study, we identified a taxol derivative named 7-epi-10-deacetyltaxol (EDT) from the culture of an endophytic fungus Pestalotiopsis microspora isolated from the bark of Taxodium mucronatum. This study was carried out to investigate the effects of fungal EDT on cell proliferation, the induction of apoptosis and the molecular mechanisms of apoptosis in human hepatoma HepG2 cells in vitro. METHODS: The endophytic fungus was identified by traditional and molecular taxonomical characterization and the fungal EDT was purified using column chromatography and confirmed by various spectroscopic and chromatographic comparisons with authentic paclitaxel. We studied the in vitro effects of EDT on HepG2 cells for parameters such as cell cycle distribution, DNA fragmentation, reactive oxygen species (ROS) generation and nuclear morphology. Further, western blot analysis was used to evaluate Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), p38-mitogen activated protein kinase (MAPK) and poly [ADP-ribose] polymerase (PARP) expression. RESULTS: We demonstrate that the fungal EDT exhibited significant in vitro cytotoxicity in HepG2 cells. We investigated cytotoxicity mechanism of EDT in HepG2 cells. The results showed nuclear condensation and DNA fragmentation were observed in cells treated with fungal EDT. Besides, the fungal EDT arrested HepG2 cells at G2/M phase of cell cycle. Furthermore, fungal EDT induced apoptosis in HepG2 cells in a dose-dependent manner associated with ROS generation and increased Bax/Bcl-2 ratio, p38 MAPKs and PARP cleavage. CONCLUSIONS: Our data show that EDT induced apoptotic cell death in HepG2 cells occurs through intrinsic pathway by generation of ROS mediated and activation of MAPK pathway. This is the first report for 7-epi-10-deacetyltaxol (EDT) isolated from a microbial source.

Splenic Epidermoid Cyst in a Five-Year-Old Child.

Splenic epidermoid cysts are rare non-parasitic true cysts affecting the spleen. We report a five-year-old child who presented with an abdominal lump associated with pain of 15 days. Ultrasonography of the abdomen showed a huge cystic lesion of obscure origin. At laprotomy a huge unilocular cyst involving upper part of spleen containing pultaceous fluid was seen and its removal necessitated splenectomy. Histopathological findings were consistent with splenic epidermoid cyst. Thus histopathology helped in elucidating the aetiology and diagnosis.

Temperature and functional plasticity of L-type Ca2+ channels in Drosophila.

The question of optimization of ion channel function to surrounding temperatures in poikilothermic organisms remains largely uninvestigated. Here, we addressed it by studying the temperature-dependence of L-type Ca2+ channels (LTCCs) in Drosophila larval muscles in the context of their modulation by protein kinase A (PKA). LTCC currents were recorded between 4 and 30°C. Different aspects of LTCC function reached maxima between 15 and 25°C: conductance, tail current amplitude, inactivation rate, and the level of basal up-regulation by PKA (26% at 21°C). Anomalous temperature-dependencies of LTCC conductance and kinetics were similar in control and in the presence of the PKA inhibitor H-89. Analysis of deactivation kinetics revealed excessive tail currents at lower temperatures (up to 15°C), indicative of voltage-dependent facilitation of LTCCs. Tail current magnitude gradually decreased with temperature from a maximum at 15°C until a nearly complete disappearance at 30°C. Elimination of excessive tail currents at higher temperatures coincided with unusual slowing of inactivation, suggesting disruption of the facilitation by rising temperature, possibly through depletion of the pool of contributing channels. Overall, these results suggest the presence of a physiological plasticity optimum of LTCC function in the temperature range of normal Drosophila development.

Inhibition of HERG potassium channels by celecoxib and its mechanism.

BACKGROUND: Celecoxib (Celebrex), a widely prescribed selective inhibitor of cyclooxygenase-2, can modulate ion channels independently of cyclooxygenase inhibition. Clinically relevant concentrations of celecoxib can affect ionic currents and alter functioning of neurons and myocytes. In particular, inhibition of Kv2.1 channels by celecoxib leads to arrhythmic beating of Drosophila heart and of rat heart cells in culture. However, the spectrum of ion channels involved in human cardiac excitability differs from that in animal models, including mammalian models, making it difficult to evaluate the relevance of these observations to humans. Our aim was to examine the effects of celecoxib on hERG and other human channels critically involved in regulating human cardiac rhythm, and to explore the mechanisms of any observed effect on the hERG channels. METHODS AND RESULTS: Celecoxib inhibited the hERG, SCN5A, KCNQ1 and KCNQ1/MinK channels expressed in HEK-293 cells with IC(50)s of 6.0 µM, 7.5 µM, 3.5 µM and 3.7 µM respectively, and the KCND3/KChiP2 channels expressed in CHO cells with an IC(50) of 10.6 µM. Analysis of celecoxib's effects on hERG channels suggested gating modification as the mechanism of drug action. CONCLUSIONS: The above channels play a significant role in drug-induced long QT syndrome (LQTS) and short QT syndrome (SQTS). Regulatory guidelines require that all new drugs under development be tested for effects on the hERG channel prior to first administration in humans. Our observations raise the question of celecoxib's potential to induce cardiac arrhythmias or other channel related adverse effects, and make a case for examining such possibilities.

Skeletal metastasis in gall bladder cancer.

This report describes an interesting and unusual case of carcinoma gallbladder with skull metastasis.

Modulation of L-type calcium channels in Drosophila via a pituitary adenylyl cyclase-activating polypeptide (PACAP)-mediated pathway.

Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca(2+) channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. We now present data suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6-38, an antagonist at type-I PACAP receptor. 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2',5'-dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca(2+) channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila.