Elemental mapping of fluorine using electron spectroscopic imaging for the intracellular localisation of an anthracycline drug (ME2303) in human ovarian cancer cells.

A major limitation to the clinical usefulness of cancer chemotherapy is the onset of acquired drug resistance, in which the effectiveness of a drug such as Doxorubicin (DOX), used in a wide variety of neoplasms, diminishes following repeated administration. Resistance is associated with drug exclusion from tumour cell nuclei. New analogues of DOX have been synthesised to reduce patient cardiotoxicity and to increase anti-tumour activity. More recently, a 2-fluoroglycoside of DOX (ME2303) has been shown to be more resistant to glycolysis and has marked anti-proliferative effects on DOX-resistant tumours. The aim of the current study was therefore to determine the intracellular localisation of ME2303 in drug sensitive and resistant human ovarian cancer cell aggregates by mapping fluorine, as a means of understanding the complex mechanisms of drug resistance. Cell aggregates of the human ovarian cell line A2780 and its DOX-resistant subline 2780AD were cultured for 1hr in 5μM of the drug ME2303, then chemically fixed and embedded in Lowicryl K4M using alcohol dehydration at ?20°C. Ultrathin sections (40-50nm) were examined on a Zeiss TEM 902 energy filtering electron microscope using 80Kv at a magnification of 12,000x. Fluorine maps were generated using the two window method by collecting two integrated images above and two integrated images below the K ionisation edge for fluorine (685eV) with a 15eV window. Image sequences were collected within 20sec to minimise the effects of mass loss from the specimen via a Dage SIT 66 video camera connected to a DT 2861 video interface board (512x512 pixels with 256 grey levels) within a 486 PC. In the A2780 cell line, fluorine was found to be distributed diffusely within the cytoplasm and at discrete sites within the nucleus. In contrast, fluorine in the 2780AD subline (co-cultured with 1.4μM DOX to maintain resistance) was found to be largely associated with the peri-nuclear Golgi region and with mitochondria, but was also found within cell nuclei, along the inner nuclear envelope and in the nucleolus. The intra-nuclear localisation of fluorine suggests that even in the presence of DOX, ME2303 can mediate anti-proliferative activity in DOX resistant human ovarian cancer cells by effective nuclear translocation.     

Biosynthesis and incorporation into protein of norleucine by Escherichia coli

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.     

Popliteal-based filleted lower leg musculocutaneous free-flap coverage of a hemipelvectomy defect.

A 33-year-old man suffered from locally recurrent malignant fibrous histiocytoma of his left thigh unresponsive to previous excision, radiation therapy, chemotherapy, and hyperthermic treatment. He underwent radical hemipelvectomy for cure. Because of extensive tumor involvement, a free flap consisting of his distal left leg based on the popliteal artery was utilized to close the defect. Both the tibia and fibula were removed from their periosteal sheaths, and the foot was excised from the flap. The popliteal artery and vein were anastomosed to the iliac vessels. The flap survived, and the patient was discharged home after physical rehabilitation. We suggest that uninvolved portions of the distal leg may be utilized as a free flap to successfully close hemipelvectomy defects in selected patients when conventional pedicle flaps are unavailable.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.     

The effect of monovalent cations on the catalytic activity of thrombin

The effect of monovalent cations on the catalytic action of thrombin has been examined utilizing a variety of substrates. Sodium chloride noncompetitively inhibited the action of thrombin on α-tosyl-l-arginine methyl ester and α-N-benzoyl-l-arginine-p-nitroanilide. No inhibition was noted when α-N-benzoyl-l-arginine ethyl ester was the substrate. The extent of inhibition was considerably less with either potassium chloride or lithium chloride. The rate of inactivation of thrombin by 1-chloro-3-tosylamido-7-amino-l-2-heptanone was reduced in the presence of sodium ions. The results are interpreted to show a specific effect of sodium ions on the ability of the active-site histidine residue to participate in thrombic catalysis.