Adenylyl cyclase, a coincidence detector for nitric oxide.

Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the Vmax of the enzyme without altering the Km for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.     

MTT assay based inhibition analysis of A2780 cells proliferation using Mollugo nudicaulis Lam. extract with CXCR4 and HER2 expression

It is of interest to document the inhibition of A2780 cell proliferation using Mollugo nudicaulis Lam.(M.nudicaulis) extract by MTT assay and by monitoring the CXCR4 and HER2 expression through RT-PCR analysis. Results shown that the n-hexane extract of M.nudicaulis have anticancer activity IC₅₀ values of 32.46±0.92 µg/mL on A2780 cell lines. It is further found that the CXCR4 and HER2 mRNA and protein expression were significantly reduced in M.nudicaulis treated A2780 cell lines. Thus, the n-hexane extract of M.nudicaulis is a natural source of bioactive compounds as potential anticancer agents.     

Retrospective assessment of clonal origin of cell lines

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.     

Use of a communal roost by Turkey Vultures in northeastern Iowa

ABSTRACT.   Communal roosts are an important aspect of Turkey Vulture ( Cathartes aura ) biology, but remain inadequately studied. We observed the use patterns of Turkey Vultures at a communal roost in northeastern Iowa from their arrival on 17 March 2005 to their departure on 19 October 2005. The roost was on a forested hillside and vultures roosted below the forest canopy in 10 live deciduous trees, spending an estimated 10–16 h per day in the roost. The birds also used nearby pre- and postroost perch sites, and formed pre- and postroost kettles near the roost site. The number of vultures using the roost ranged from about 20 early in the season to a peak of 281 on 4 October. The apparent pattern was one of moderate and fluctuating numbers early in the 7-month season, increasing numbers in the middle of the season, and high and fluctuating numbers late in the season. Hatching-year vultures began to appear at the roost during the last week of August. The mean monthly time of departure from the roost ranged from 3 min before sunrise to 131 min after sunrise. Vultures departed the roost significantly later in the morning during the summer (June, July, and August) than during other months (April, May, September, and October), probably due to differences in soaring conditions (longer thermal generation times during the summer) and available foraging time (longer days in the summer).