The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

The stable inheritance of the 2μm plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2μm circle-derived plasmid shows relatively poor stability.     

Establishment of an in vitro assay for assessing the effects of drugs on the liver stages of Plasmodium vivax malaria

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.     

Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.     

A novel disintegrin protein from Naja naja venom induces cytotoxicity and apoptosis in human cancer cell lines in vitro

Animal venoms and toxins are potential bioresources that have been known to mankind as a therapeutic tool for more than a century through folk and traditional medicine. The purified “disintegrin protein” (64 kDa) from the venom of the Indian cobra snake (Naja naja) exhibited cytotoxic effects of various types of human cancer cell lines such as breast cancer (MCF-7), lung cancer (A549) and liver cancer (HepG2). In vitro cytotoxicity, DNA fragmentation, an apoptotic assay and a cell cycle analysis were performed to evaluate the anticancer activity of disintegrin against the above cell lines. The IC₅₀ value of disintegrin was determined to be 2.5 ± 0.5 μg/mL, 3.5 ± 0.5 μg/mL, and 3 ± 0.5 μg/mL for the MCF-7, A549 and HepG2 cell lines respectively. Moreover, the increased distribution of G0/G1 and S phase led to decreased populations of cells in the G2/M phase of MCF-7, HepG2 and A549 cells.     

Chronic toxicity bioassay and effect of endosulfan,HCH, copper and zinc on the survival and intermoult duration of larvae of mangrove crab Macrophthalmus erato De Man

The effects of insecticides (endosulfan, HCH) and heavy metals (copper and zinc) were studied on survival and duration of the moult cycle of larvae and megalopa of a common mangrove crab, Macrophthalmus erato, that inhabits Pitchavaram mangroves, southeast coast of India. In chronic toxicity test, percentage of survival from hatchling to megalopa stage at different concentrations of endosulfan (0.048 to 0.36 microg/L), HCH (0.32 to 2.4 microg/L), copper (9.0 to 67.5 microg/L) and zinc (15.2 to 139.0 microg/L) were 6, 12, 8 and 12 respectively. Endosulfan, HCH, copper and zinc considerably increased the total period in development from first zoea to megalopa stage from 18 days in the control to 23, 21, 20 and 22 days in the treated specimens. It is concluded that concentrations well below the observed chronic concentrations, may prove to be more deleterious to larva of this species in natural environment leading to reduction in the success of recruitment to the adult population.     

Effect of feed on larvae of Macrobrachium malcolmsonii (H. Milne Edwards) reared in synthetic brackishwater

Freshwater prawn, Macrobrachium malcolmsonii, has potential for aquaculture. Larvae of M. malcolmsonii were reared in synthetic brackishwater using commercial salts. Live and artificial feeds were provided to larvae either individually or in combination. Survival was significantly higher when larvae were fed with live feed only. However, larvae fed with artificial feed showed significantly lower survival. Survival was significantly higher when Artemia combined together with other feeds. Despite its limitations, synthetic brackishwater can be used for freshwater prawn seed production.     

Spatial distribution of nematode genera in relation to host plants of Western Ghats,Tamil Nadu,India

A survey of plant parasitic nematodes (PPN) abundance and diversity was conducted in foothills of Western Ghats at Coimbatore and Nilgiris districts in Tamil Nadu, India. Our study pointed to evaluate the PPN association with weeds, shrubs, and herbs including some agricultural crop cover both agro and forest ecosystems. Soil samples were collected around the rhizosphere of the plants and nematodes were isolated, counted under a microscope and identified by morphological characters. We collected 415 soil samples around the rhizosphere of 84 major plant families. Among 84 plant families, we found 13 PPN genera those generally responsible for cash crop yield loss. The genus Helicotylenchus spp., and Meloidogyne spp., were found frequent (226,136 samples) and rarely Hemicycliophora spp.,. The PPN diversity was measured by Shannon diversity index which showed that the diversity of PPN found higher in plant species those from the families Rosaceae (2.20) and Rubiaceae (2.13). We also recorded the PPN richness was dominated in four plant families. This result reveals that the PPN diversity and richness are positively correlated with host plant species diversity and altitude. Further, our study concludes that a wild plant in the forest ecosystem behaves like a reservoir of PPN.     

Further evidence for BRCA1 communication with the inactive X chromosome

BRCA1, a breast and ovarian cancer-suppressor gene, exerts tumor-suppressing functions that appear to be associated, at least in part, with its DNA repair, checkpoint, and mitotic regulatory activities. Earlier work from our laboratory also suggested an ability of BRCA1 to communicate with the inactive X chromosome (Xi) in female somatic cells (Ganesan et al., 2002). Xiao et al. (2007) (this issue of Cell) have challenged this conclusion. Here we discuss recently published data from our laboratory and others and present new results that, together, provide further support for a role of BRCA1 in the regulation of XIST concentration on Xi in somatic cells.