The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Molecular analysis of gene expression in the developing pontocerebellar projection system

As an approach toward understanding the molecular mechanisms of neuronal differentiation, we utilized DNA microarrays to elucidate global patterns of gene expression during pontocerebellar development. Through this analysis, we identified groups of genes specific to neuronal precursor cells, associated with axon outgrowth, and regulated in response to contact with synaptic target cells. In the cerebellum, we identified a phase of granule cell differentiation that is independent of interactions with other cerebellar cell types. Analysis of pontine gene expression revealed that distinct programs of gene expression, correlated with axon outgrowth and synapse formation, can be decoupled and are likely influenced by different cells in the cerebellar target environment. Our approach provides insight into the genetic programs underlying the differentiation of specific cell types in the pontocerebellar projection system.     

Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes.     

Comparison of responses elicited by immunization with a Legionella species common lipoprotein delivered as naked DNA or recombinant protein

To evaluate the peptidoglycan-associated lipoprotein (PAL) antigen of Legionella pneumophila as a vaccine candidate, mice were immunized intramuscularly with pcDNA3-PAL and intraperitoneally with recombinant PAL (t-rPAL), which were compared for their ability to induce PAL-specific immune responses. The t-rPAL protein induced PAL-specific IgG antibody production significantly more than did pcDNA3-PAL. The IgG2a and IgG1 production was predominant after pcDNA3-PAL and t-rPAL administration, respectively. In particular, pcDNA3-PAL induced much higher PAL-specific cytotoxic T-lymphocyte responses than did t-rPAL. Furthermore, in vivo, CD19+ B-cell populations were dramatically increased by t-rPAL vaccination, suggesting a B-cell immunomodulatory activity of the lipoprotein. The PAL antigen was also conserved among Legionella species, as determined by PCR and immunoblot analyses. These results support a potential use of the t-rPAL protein and in particular DNA vaccines against Legionella infections.     

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

Apoptosis induction in HL-60 cells and inhibition of topoisomerase II by triterpene celastrol

Celastrol, which is a triterpene purified from Celastraceae plants, has anticancer and anti-inflammatory activities. In this study we investigated to clarify whether celastrol can induce apoptosis in a human leukemia HL-60 model system. Celastrol was found to induce apoptosis, and the rank order of the potency of celastrol and its derivatives to induce internucleosomal DNA fragmentation was found to be celastrol>cela-H>the other derivatives=vehicle control. Many anticancer agents are known to possess the ability to inhibit topoisomerase II, so the inhibitory activities of celastrol and its derivatives on topoisomerase II were also explored. The rank order of the inhibitory activity was found to be celastrol>etoposide>cela-H, indicating that the apoptosis-inducing activities of cela derivatives correspond to their inhibitory activities on topoisomerase II. These data suggested that celastrol may cause its effects such as anticancer activity by the mechanism of apoptosis along with topoisomerase II inhibition.     

White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines

The prevalence and geographic distribution of white spot syndrome virus (WSSV) infection among cultured penaeid shrimp in the Philippines was determined from January to May, 1999, using PCR (polymerase chain reaction) protocol and Western blot assays. A total of 71 samples consisting of 18 post-larvae (PL) and 53 juvenile/adult shrimp samples (56 to 150 days-of-culture, DOC) were screened for WSSV. Of the 71 samples tested, 51 (72%) were found positive for WSSV by PCR: 61% (31/51) after 1-step PCR and 39% (20/51) after 2-step, non-nested PCR. Of the PL and juvenile/adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. By Western blot, only 6 of the 51 (12%) PCR-positive samples tested positive for WSSV. Of the 20 samples negative for WSSV by PCR, all tested negative for WSSV by Western blot assay. This is the first report of the occurrence of WSSV in the Philippines.     

Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4-chlorophenol in the presence of a conventional carbon source

Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data.     

Amplified fragment length polymorphism fingerprinting of 16 banana cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

The Saccharomyces cerevisiae YCC5 (YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine

The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.