Response of nasopharyngeal carcinoma cells to Epstein-Barr virus infection in vitro

Many nasopharyngeal carcinoma (NPC) biopsy specimens contain Epstein-Barr virus (EBV). However, the response of NPC cells to EBV infection in vitro and in vivo is not well characterized. In this experiment we infected NPC cells with EBV particles through endocytosis of a complex of EBV immunoglobulin A (IgA) secretory component (SC) protein to observe the response of host cells to the foreign viral infection in vitro. We found that EBV particles were endocytosed and stabilized in NPC nuclei 24 hours after infection; the EBV genomes were then gradually decreased after serial passages within 3 to 4 weeks by the following pathway: the EBV genomes first moved toward the nuclear envelope from the center of the nucleus; after crossing the nuclear envelope, they moved into the cytoplasm and toward the plasma membrane and were discharged by exocytosis. At the 10th day of EBV infection, EBV-latent membrane protein-1 and Epstein-Barr nuclear antigen (EBNA)-1 protein expressions could be detected, but not EBV-viral capsid antigen. Observation of EBNA-1 protein and host growth factor and cytokine gene expressions in the weeks after incubation revealed that the EBNA-1 protein expression was decreased proportionally with decrease of EBV genome. The mRNA expression of epithelial growth factor receptor, transforming growth factor (TGF)-alpha, interleukin (IL)-1beta, IL-6, and granulocyte-macrophage colony-stimulating factor increased within 1 to 2 weeks after infection, and gradually recovered to the original level at 3 to 4 weeks, whereas the mRNAs of TGFbeta1, TGFbeta receptor type I (TGFbetaRI), TGFbetaR type II, IL-8, and tumor necrosis factor-alpha remained unchanged. It is concluded that in vitro EBV infection in NPC cells results in increase of certain growth factor and cytokine gene expressions in host cells. The change in gene expression returns to the original level approximately 3 to 4 weeks after infection because of exocytosis of EBV DNA by the infected cells through an unidentified mechanism.     

Loss of CD8 and TCR binding to Class I MHC ligands following T cell activation

The capacity of T cells to bind peptide/MHC ligands changes with T cell development and differentiation. Here we study changes in peptide/MHC multimer binding following T cell activation. Surprisingly, T cell activation caused a marked reduction in specific peptide/MHC Class I multimer binding, which was distinct from transient TCR down-regulation, and was especially dramatic for engagement with low-affinity peptide/MHC ligands. Direct CD8-Class I interactions were also profoundly and rapidly impaired following T cell stimulation, even though surface CD8alpha and CD8beta levels were unchanged after activation, suggesting that decreased CD8 co-receptor binding contributes to this effect. Finally, we show that enzymatic desialylation restores much of the multimer binding on activated T cells, suggesting that altered glycosylation may inhibit TCR/CD8 binding to peptide/MHC ligands. These radical changes in activated T cells' ability to perceive peptide/MHC ligands may contribute to selective outgrowth of clones with high affinity for the stimulatory ligand.     

Influence of vascular parameters on the effectiveness of intra-aortic balloon pumping: A model study

The intra-aortic balloon pump has been widely used as a temporary heart-assist device. In this investigation, a nonlinear mathematical model of the arterial system and intra-aortic balloon pump was studied analytically. Thus, the influences of a number of vascular parameters on the effectiveness of intra-aortic balloon pumping (IABP) were determined. The effects of changes in vascular parameters of the model on a number of performance indexes were investigated. These performance indexes (aortic mean diastolic pressure, aortic end diastolic pressure, cardiac output, coronary flow and phase differences between the fundamental Fourier components of aortic root pressure and flow) were used as the criterion for an evaluation of the effectiveness of the assist pump. The following vascular parameters were perturbed by four steps (±10%, ±20%) from the values in the standard model: heart rate, peripheral resistance, left ventricular pressure, aortic elastance, aortic radius, arterial wall thickness, and aortic length. This model was evaluated for a wide range of balloon-pump phase-control settings (assisted case) and for the unassisted case (when the pump is disabled). It is concluded that changes in heart rate, peripheral resistance and left ventricular pressure cause the most significant changes in pump performance. Dr. Ohley is with the Datascope Corporation Dr. Kao is with the Technicare Corporation     

A protein kinase A-dependent molecular switch in synapsins regulates neurite outgrowth

Cyclic AMP (cAMP) promotes neurite outgrowth in a variety of neuronal cell lines through the activation of protein kinase A (PKA). We show here, using both Xenopus laevis embryonic neuronal culture and intact X. laevis embryos, that the nerve growth-promoting action of cAMP/PKA is mediated in part by the phosphorylation of synapsins at a single amino acid residue. Expression of a mutated form of synapsin that prevents phosphorylation at this site, or introduction of phospho-specific antibodies directed against this site, decreased basal and dibutyryl cAMP-stimulated neurite outgrowth. Expression of a mutation mimicking constitutive phosphorylation at this site increased neurite outgrowth, both under basal conditions and in the presence of a PKA inhibitor. These results provide a potential molecular approach for stimulating neuron regeneration, after injury and in neurodegenerative diseases.     

Construction and characterization of three region-specific microdissection libraries for human chromosome 18

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and MboI linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q 11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50–600 bp, with a mean of 180–220 bp for the three libraries. The libraries contained approximately 40–60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human gemomic HindIII fragments hybridized to each microclone were determined and microclones crosshybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating hughly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.     

Actions of some anions on electrical properties and mechanical threshold of frog twitch muscle

1. Using two micro-electrodes in a point-voltage clamp technique, the effects of the lyotropic anions, NO(3) (-), Br(-), I(-), SCN(-), and CH(3)SO(4) (-), and of SO(4) (2-) on the mechanical threshold and electrical properties of frog sartorius muscle were studied.2. In chloride Ringer solution the spike threshold was -59 mV, mechanical threshold -48 mV, and the threshold for delayed rectification of the total current at about 100 msec -52 mV.3. When Cl(-) was replaced by one of the lyotropic anions, the effective resistance determined at -100 mV tended to increase. But, because of the variability of the effective resistance in individual fibres, most lyotropic anions did not cause a statistically significant increase in the effective resistance. Only I(-) and SO(4) (2-) significantly increased the effective resistance.4. Most lyotropic anions had no significant effect on the spike threshold; I(-), at 58 mM, lowered it slightly. Sulphate raised the threshold.5. Tetrodotoxin (0.1 mug/ml.) abolished the spikes, but did not affect the mechanical and delayed rectification thresholds. It was, therefore, used to pre-treat all preparations for determining these thresholds.6. All lyotropic anions lowered the mechanical and the delayed rectification thresholds, the order of effectiveness being approximately SCN(-) > I(-) > NO(3) (-) > CH(3)SO(4) (-) > Br(-). As in Cl(-) Ringer, the two thresholds lay very close together in every case. Sulphate raised slightly both the mechanical and delayed rectification thresholds, again in close parallel.7. This close agreement of the mechanical and delayed rectification thresholds is not caused by movement artifact, because in fibres in which visible contractions were eliminated with hypertonic solutions the delayed rectification thresholds were the same as those in contracting fibres.8. In spite of the close agreement, reasons are given to doubt a direct causal relationship between the mechanical and delayed rectification thresholds.9. Nitrate apparently had little effect on the rate of inactivation of the outward current, or on the relation between steady-state inactivation and membrane potential.     

Genetic predetermination of quantitative expression of HLA antigens in platelets and mononuclear leukocytes

In view of the potential functional importance of quantitative expression of HLA antigens, a series of studies were conducted to determine the relative quantities of specific HLA-A and -B antigens expressed in MNLs and platelets of HLA-phenotyped family members and unrelated individuals. An mAb that reacts with a well-defined monomorphic epitope in the α₃ domain of the heavy chains of HLA molecules was developed and used to quantify each HLA-A or -B antigen on western blots of IEF gels. The results of these studies demonstrated that the relative quantities of HLA-A and -B antigens in platelets and MNLs of an individual did not change over time. Further studies showed that the relative quantities of HLA-A and -B antigens for haplotypes shared among the first-degree relatives were always the same and followed Mendelian inheritance. In contrast, the relative quantities of HLA-A and -B antigens for a haplotype shared by unrelated individuals varied significantly. All these findings support the hypothesis that the quantitative expression of HLA antigens is genetically predetermined and may play important roles in determining disease susceptibility and severity. Human Immunology 38, 243–250 (1993)     

Genome-wide profiling of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a common malignancy, the incidence of which is particularly high in some Asian countries due to the geographically linked areca quid (AQ) chewing habit. In this study, array-based comparative genomic hybridization was used to screen microdissected OSCCs for genome-wide alterations. The highest frequencies of gene gain were detected for TP63, Serpine1, FGF4/FGF3, c-Myc and DMD. The highest frequencies of deletion were detected for Caspase8 and MTAP. Gained genes, classified by hierarchical clustering, were mainly on 17q21-tel; 20q; 11q13; 3q27-29 and the X chromosome. Among these, gains of EGFR at 7p, FGF4/FGF3, CCND1 and EMS1 at 11q13, and AIB1 at 20q were significantly associated with lymph node metastasis. The genomic profiles of FHIT and EXT1 in AQ-associated and non-AQ-associated OSCCs exhibited the most prominent differences. RT-PCR confirmed the significant increase of TP63 and Serpine1 mRNA expression in OSCC relative to non-malignant matched tissue. A significant increase in Serpine1 immunoreactivity was observed from non-malignant matched tissue to OSCC. However, there was no correlation between the frequent genomic loss of Caspase 8 and a significant decrease in Caspase8 expression. These data demonstrate that genomic profiling can be useful in analysing pathogenetic events involved in the genesis or progression of OSCC.