Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus interacts with origin recognition complexes at the LANA binding sequence within the terminal repeats

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.     

Simulation of turbulent airflow using a CT based upper airway model of a racehorse

Computational model for airflow through the upper airway of a horse was developed. Previous flow models for human airway do not hold true for horses due to significant differences in anatomy and the high Reynolds number of flow in the equine airway. Moreover, models that simulate the entire respiratory cycle and emphasize on pressures inside the airway in relation to various anatomical diseases are lacking. The geometry of the airway was created by reconstructing images obtained from computed tomography scans of a thoroughbred racehorse. Different geometries for inhalation and exhalation were used for the model based on the difference in the nasopharynx size during the two phases of respiration. The Reynolds averaged Navier-Stokes equations were solved for the isothermal flow with the standard k-epsilon model for turbulence. Transient pressure boundary conditions for the entire breathing cycle were obtained from past experimental studies on live horses. The flow equations were solved in a commercial finite volume solver. The flow rates, computed based on the applied pressure conditions, were compared to experimentally measured flow rates for model validation. Detailed analysis of velocity, pressure, and turbulence characteristics of the flow was done. Velocity magnitudes at various slices during inhalation were found to be higher than corresponding velocity magnitudes during exhalation. The front and middle parts of the nasopharynx were found to have minimum intraluminal pressure in the airway during inhalation. During exhalation, the pressures in the soft palate were higher compared to those in the larynx, epiglottis, and nasopharynx. Turbulent kinetic energy was found to be maximum at the entry to the airway and gradually decreased as the flow moved inside the airway. However, turbulent kinetic energy increased in regions of the airway with abrupt change in area. Based on the analysis of pressure distribution at different sections of the airway, it was concluded that the front part of the nasopharynx requires maximum muscular activity to support it during inhalation. During exhalation, the soft palate is susceptible to displacements due to presence of high pressures. These can serve as critical information for diagnosis and treatment planning of diseases known to affect the soft palate and nasopharynx in horses, and can potentially be useful for human beings.     

Modeling spinal muscular atrophy in Drosophila

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets.     

Changes in polyamine contents during root and nodule growth of Phaseolus mungo

In Phaseolus mungo seeds, polyamine content increased during early germination, being maximum after 24 hr; and the arginine decarboxylase showed a peak after 18 hr. During nodule initiation and growth two peaks of polyamine contents were noted-the first being 2 weeks after nodule initiation and a second one after 5 weeks. Arginine decarboxylase activity also followed the same pattern. In the roots the polyamine concentration as well as arginine decarboxylase increased up to week 2 after sowing followed by a gradual decrease. Estimation of RNA, DNA and protein contents showed a pattern similar to that of the polyamines.     

32P uptake in three submerged aquatic plant species

Summary Highest uptake of³²P by young shoots of three plant species was observed and lowest by old ones. The uptake of³²P was highest inHydrilla shoots, followed byVallisneria andPotamogeton.Kinetin (0.23 mM) pretreatment (24 h) increased the uptake of³²P, while 0.69 mM ethrel or 0.075 mM ABA decreased it in all species.³²P was transported to the largest extent to the young shoots of the submerged plants and to the smallest extent to the old ones by kinetin pretreatment. Kinetin enhanced the uptake of³²P most inHydrilla shoots, followed byVallisneria andPotamogeton. Ethrel diminished³²P uptake most inPotamogeton shoots and to the smallest extent inHydrilla, while ABA lowered it most inHydrilla shoots and to the smallest extent inPotamogeton. Kinetin, ethrel and ABA can modify the uptake of³²P of these aquatic plants.     

Changes in the Activities of Ribulose 1,5-Bisphosphate and Phosphoenolpyruvate Carboxylases in Submersed Aquatic Angiosperms during Aging

Changes in the activities of ribulose-1,5-bisphosphate carboxylase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPcase) during aging and senescence of leaves of submersed aquatic angiosperms, Potamogeton pectinatus L., Vallisneria spiralis L., and Hydrilla verticillata (L.f.) Royle were studied. The activity of RuBPcase decreased with both aging and senescence in three species. PEPcase activity increased from young to mature leaves in all three plants and also in old leaves of Hydrilla. The ratio of RuBPcase to PEPcase activity was lowest in mature and highest in old leaves, and increased with aging of isolated mature leaves in Potamogeton and Vallisneria, but decreased markedly in Hydrilla with aging and senescence. Kinetin treatment (0.23 mm) increased RuBPcase activity in three species and PEPcase activity in Potamogeton and Vallisneria, but decreased PEPcase activity in Hydrilla. Treatments with 2-chloroethylphosphonic acid (Ethrel, 0.69 mm) and abscisic acid (ABA, 0.075 mm) showed almost an opposite trend.     

Failure in peripheral immuno-surveillance due to thymic atrophy: importance of thymocyte maturation and apoptosis in adult tumor-bearer

Tumor-induced immunosuppression often leads to failure in cancer therapy. Here, in an attempt to understand the course of tumor-dependent immunosuppression in young adult murine model, we found that in Ehrlich's ascites carcinoma (EAC) bearing mice, CD4(+) and CD8(+) populations of peripheral blood were depleted within first week of tumor inoculation. However, there was a rise in these populations at the end of second week only to fall back severely at the end of third week. These pulsating changes were also reflected in spleen. Interestingly, in thymus, production of CD4(+) and CD8(+) increased during first two weeks of tumor inoculation indicating the effort of thymus to replenish these populations in peripheral blood and spleen in response to their initial depletion, restricting tumor growth in between first and second weeks. However, at third week, due to (a) block in thymocyte maturation leading to increase in CD4(-)8(-) and decrease in CD4(+)8(+), (b) inhibition in formation of functional isotypes, and (c) thymocyte apoptosis, thymic reinforcement was stalled. Further investigation for the underlying mechanism of such thymic atrophy revealed down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, resulting in decreased Bcl-2/Bax ratio thereby inducing apoptosis. Above findings accounted for the significant decrease in CD4(+) and CD8(+) of peripheral blood and spleen by the end of third week culminating in total collapse in the fight back mechanism of host and uncontrolled growth of tumor. All these results signify the importance of thymus in modulating the immune status of the host during tumor development.     

Immunology: How Do T Cells Recognize Antigen?

T cells recognize small fragments of microorganisms (antigens) on the surface of other cells using T cell antigen receptors. The mechanism by which these receptors signal into T cells is controversial, but two recent studies provide important new clues.