Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.     

Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers

The eukaryotic family of ADP-ribosylation factor (Arf) GTPases plays a key role in the regulation of protein trafficking, and guanine-nucleotide exchange is crucial for Arf function. Exchange is stimulated by members of another family of proteins characterized by a 200-amino acid Sec7 domain, which alone is sufficient to catalyze exchange on Arf. Here, we analyzed the phylogeny of Sec7-domain-containing proteins in seven model organisms, representing fungi, plants, and animals. The phylogenetic tree has seven main groups, of which two include members from all seven model systems. Three groups are specific for animals, whereas two are specific for fungi. Based on this grouping, we propose a phylogenetically consistent set of names for members of the Sec7-domain family. Each group, except for one, contains proteins with known Arf exchange activity, implying that all members of this family have this activity. Contrary to the current convention, the sensitivity of Arf exchange activity to the inhibitor brefeldin A probably cannot be predicted by group membership. Multiple alignment reveals group-specific domains outside the Sec7 domain and a set of highly conserved amino acids within it. Determination of the importance of these conserved elements in Arf exchange activity and other cellular functions is now possible.     

Placental protein 14 regulates selective B cell responses

Placental protein 14 (PP14) is a glycoprotein of the lipocalin family that acts as a negative regulator in T cell receptor-mediated activation. In this study, we investigated PP14s potential role in regulating B cell activation. While PP14-inhibited B cell proliferation, IgM secretion and the surface expression of MHC class II, the expression of other surface molecules, such as CD69 and CD86, were unaffected. These observed effects were independent of the anti-IgM concentration used for stimulation, regardless of the presence of either T cells or IL-4, and persisted when B cells were stimulated by stimuli, which circumvent early events during B cell Ag receptor (BCR) activation, namely, protein kinase C activators in combination with Ca(2+) ionophore. Interestingly, we demonstrated that PP14s inhibitory characteristics are reminiscence of that achieved by independent ligation of CD19 using anti-CD19 mAb. Together with our previously reported effects on T cells, these findings identify PP14 as a soluble regulatory factor capable of interacting with both T and B cells in a carbohydrate-dependent manner and as a result it can affect both cellular and humoral immune responses.     

How reliable are experimental protein-protein interaction data?

Data of protein-protein interactions provide valuable insight into the molecular networks underlying a living cell. However, their accuracy is often questioned, calling for a rigorous assessment of their reliability. The computation offered here provides an intelligible mean to assess directly the rate of true positives in a data set of experimentally determined interacting protein pairs. We show that the reliability of high-throughput yeast two-hybrid assays is about 50%, and that the size of the yeast interactome is estimated to be 10,000-16,600 interactions.     

Utilizing logical relationships in genomic data to decipher cellular processes

The wealth of available genomic data has spawned a corresponding interest in computational methods that can impart biological meaning and context to these experiments. Traditional computational methods have drawn relationships between pairs of proteins or genes based on notions of equality or similarity between their patterns of occurrence or behavior. For example, two genes displaying similar variation in expression, over a number of experiments, may be predicted to be functionally related. We have introduced a natural extension of these approaches, instead identifying logical relationships involving triplets of proteins. Triplets provide for various discrete kinds of logic relationships, leading to detailed inferences about biological associations. For instance, a protein C might be encoded within an organism if, and only if, two other proteins A and B are also both encoded within the organism, thus suggesting that gene C is functionally related to genes A and B. The method has been applied fruitfully to both phylogenetic and microarray expression data, and has been used to associate logical combinations of protein activity with disease state phenotypes, revealing previously unknown ternary relationships among proteins, and illustrating the inherent complexities that arise in biological data.     

Focal localization of placental protein 14 toward sites of TCR engagement

TCR signal transduction is amplified by the dynamic accumulation of accessory molecules at APC-T cell contact sites, along with the simultaneous exclusion from these sites of negative regulators, such as certain tyrosine phosphatases and large glycosylated proteins. However, given the general nature of the cytoskeleton-driven clustering mechanism underlying molecular segregation events at the APC-T cell interaction site, the possibility exists that negative regulators might similarly be segregated at these sites. Using fluorescence microscopy, we have demonstrated that placental protein 14 (PP14), a direct T cell inhibitor, focuses toward APC-T cell contact sites in conjunction with conjugate formation. We have further established that the function of PP14 is dependent upon its localization to the sites of TCR triggering, where it negatively regulates T cell activation. Thus, PP14 provides an example of a soluble negative T cell regulator whose inhibitory activity is linked to modulation of the APC-T cell contact site, thereby hindering early events triggered by the TCR.     

Replication and/or separation of some human telomeres is delayed beyond S-phase in pre-senescent cells

Cultured primary human cells, which lack telomerase, enter a state of replicative senescence after a characteristic number of population doublings. During this process telomeres shorten to a critical length of approximately 5-7 kb. The mechanistic relationship between advanced cell passage, cellular senescence and telomeric function has yet to be fully elucidated. In the study described here, we investigated the relationship between changes in telomeric replication timing and/or sister chromatid separation at telomeric regions and advanced cell passage. Using fluorescence in situ hybridization, we analyzed the appearance of double hybridization signals (doublets), which indicate that the region of interest has replicated and the replicated products have separated sufficiently to be resolved as two distinct signals. The results showed that the replication and separation of several telomeric regions occurs during the second half of S-phase and that a delay in replication and/or separation of sister chromatids at these regions occurs in pre-senescent human fibroblasts. Surprisingly, in a significant percentage of pre-senescent cells, several telomeric regions did not hybridize as doublets even in metaphase chromosomes. This delay was not associated with extensive changes in methylation levels at subtelomeric regions and was circumvented in human fibroblasts expressing ectopic telomerase. We propose that incomplete replication and/or separation of telomeric regions in metaphase may be associated with proliferative arrest of senescent cells. This cell growth arrest may result from the activation of a mitotic checkpoint, or from chromosomal instability consequent to progression in the cell cycle despite failure to replicate and/or separate these regions completely.     

Subthreshold Voltage Noise Due to Channel Fluctuations in Active Neuronal Membranes

Voltage-gated ion channels in neuronal membranes fluctuate randomly between different conformational states due to thermal agitation. Fluctuations between conducting and nonconducting states give rise to noisy membrane currents and subthreshold voltage fluctuations and may contribute to variability in spike timing. Here we study subthreshold voltage fluctuations due to active voltage-gated Na⁺ and K⁺ channels as predicted by two commonly used kinetic schemes: the Mainen et al. (1995) (MJHS) kinetic scheme, which has been used to model dendritic channels in cortical neurons, and the classical Hodgkin-Huxley (1952) (HH) kinetic scheme for the squid giant axon. We compute the magnitudes, amplitude distributions, and power spectral densities of the voltage noise in isopotential membrane patches predicted by these kinetic schemes. For both schemes, noise magnitudes increase rapidly with depolarization from rest. Noise is larger for smaller patch areas but is smaller for increased model temperatures. We contrast the results from Monte Carlo simulations of the stochastic nonlinear kinetic schemes with analytical, closed-form expressions derived using passive and quasi-active linear approximations to the kinetic schemes. For all subthreshold voltage ranges, the quasi-active linearized approximation is accurate within 8% and may thus be used in large-scale simulations of realistic neuronal geometries.     

Modeling the electrical behavior of anatomically complex neurons using a network analysis program: Passive membrane

We describe the application of a popular and widely available electrical circuit simulation program called SPICE to modeling the electrical behavior of neurons with passive membrane properties and arbitrarily complex dendritic trees. Transient responses may be calculated at any location in the cell model following current, voltage or conductance perturbations at any point. A numbering method is described for binary trees which is helpful in transforming complex dendritic structures into a coded list of short cylindrical dendritic segments suitable for input to SPICE. Individual segments are modeled as isopotential compartments comprised of a parallel resistor and capacitor, representing the transmembrane impedance, in series with one or two core resistors. Synaptic current is modeled by a current source controlled by the local membrane potential and an alpha-shaped voltage, thus simulating a conductance change in series with a driving potential. Extensively branched test cell circuits were constructed which satisfied the equivalent cylinder constraints (Rall 1959). These model neurons were perturbed by independent current sources and by synaptic currents. Responses calculated by SPICE are compared with analytical results. With appropriately chosen model parameters, extremely accurate transient calculations may be obtained. Details of the SPICE circuit elements are presented, along with illustrative examples sufficient to allow implementation of passive nerve cell models on a number of common computers. Methods for modeling excitable membrane are presented in the companion paper (Bunow et al. 1985).