Activation changes the spectrum but not the diversity of genes expressed by T cells

During activation T cells are thought to change their patterns of gene expression dramatically. To find out whether this is true for T cells activated in animals, the patterns of genes expressed in resting T cells and T cells 8 and 48 hr after activation were examined by using Affymetrix gene arrays. Gene arrays gave accurate comparisons of gene expression in the different cell types because the expression of genes known to vary during activation changed as expected. Of the approximately 6,300 genes assessed by the arrays, about one-third were expressed to appreciable extents in any of the T cells tested. Thus, resting T cells express a surprisingly large diversity of genes. The patterns of gene expression changed considerably within 8 hr of T cell activation but returned to a disposition more like that of resting T cells within 48 hr of exposure to antigen. Not unexpectedly, the activated T cells expressed genes associated with cell division at higher levels than resting T cells. The resting T cells expressed a number of cytokine receptor genes and some genes thought to suppress cell division, suggesting that the state of resting T cells is not a passive failure to respond to extant external stimuli.

p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: A third member of the p53 family?

p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a sequence-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5′-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T)(T/A)GPyPyPy-3′. We have now identified, partially purified, and characterized an additional ≈40-kDa nuclear protein, p53CP (p53 competing protein), that specifically binds to the consensus p53 binding sites found in several p53 downstream target genes, including Waf-1, Gadd45, Mdm2, Bax, and RGC. The minimal sequence requirement for binding is a 14-bp motif, 5′-CTTGCTTGAACAGG-3′ [5′-C(A/T)(T/A)GPyPyPyPuPuPuC(A/T)(T/A)G-3′], which includes the central nucleotides of the typical p53 binding site with one mismatch. p53CP and p53 (complexed with antibody) showed a similar binding specificity to Waf-1 site but differences in Gadd45 and T3SF binding. Like p53, p53CP also binds both double- and single-stranded DNA oligonucleotides. Important to note, cell cycle blockers and DNA damaging reagents, which induce p53 binding activity, were found to inhibit p53CP binding in p53-positive, but not in p53-negative, cells. This finding suggested a p53-dependent coordinate regulation of p53 and p53CP in response to external stimuli. p53CP therefore could be a third member of the p53 family, in addition to p53 and p73, a newly identified p53 homolog. p53CP, if sequestering p53 from its DNA binding sites through competitive binding, may provide a novel mechanism of p53 inactivation. Alternatively, p53CP may have p53-like functions by binding and transactivating p53 downstream target genes. Cloning of the p53CP gene ultimately will resolve this issue.

Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant responses

Methyl jasmonate is a plant volatile that acts as an important cellular regulator mediating diverse developmental processes and defense responses. We have cloned the novel gene JMT encoding an S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Arabidopsis thaliana. Recombinant JMT protein expressed in Escherichia coli catalyzed the formation of methyl jasmonate from jasmonic acid with Km value of 38.5 μM. JMT RNA was not detected in young seedlings but was detected in rosettes, cauline leaves, and developing flowers. In addition, expression of the gene was induced both locally and systemically by wounding or methyl jasmonate treatment. This result suggests that JMT can perceive and respond to local and systemic signals generated by external stimuli, and that the signals may include methyl jasmonate itself. Transgenic Arabidopsis overexpressing JMT had a 3-fold elevated level of endogenous methyl jasmonate without altering jasmonic acid content. The transgenic plants exhibited constitutive expression of jasmonate-responsive genes, including VSP and PDF1.2. Furthermore, the transgenic plants showed enhanced level of resistance against the virulent fungus Botrytis cinerea. Thus, our data suggest that the jasmonic acid carboxyl methyltransferase is a key enzyme for jasmonate-regulated plant responses. Activation of JMT expression leads to production of methyl jasmonate that could act as an intracellular regulator, a diffusible intercellular signal transducer, and an airborne signal mediating intra- and interplant communications.

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory.

Cell-surface-expressed T-cell antigen-receptor zeta chain is associated with the cytoskeleton.

The T-cell antigen receptor zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. zeta chain can associate with certain protein tyrosine kinases and retains the capacity to transduce signals independently of the other receptor subunits. Thus, zeta chain could couple cell-surface-expressed T-cell antigen receptors to the intracellular signal-transduction apparatus by its association with various intracellular molecules in addition to tyrosine kinases. In the process of searching for zeta chain-associated molecules we observed that after lysis of resting T cells with Triton X-100, zeta chain is localized in the detergent-insoluble fraction, in addition to its presence in the detergent-soluble fraction. Treatment of T cells with cytochalasin B, an actin-depolymerizing agent, leads to the complete dissociation of zeta chain from the Triton-insoluble fraction, suggesting a linkage between zeta chain and the cytoskeletal matrix. We have also determined that cytoskeletal-associated zeta chain is expressed on the cell surface. Furthermore, a tyrosine-phosphorylated 16-kDa zeta chain was detected only in the Triton-insoluble cytoskeletal fraction of resting T cells. zeta chain also maintains its association with the cytoskeleton when expressed in COS cells, inferring that the cytoskeletal elements involved in this linkage may be ubiquitous. Finally, we have localized a 42-amino acid region in the intracytoplasmic domain of zeta chain, which is crucial for maximal interaction between zeta chain and the cytoskeleton. Anchorage of cell-surface-expressed zeta chain to the cytoskeleton in resting T cells may facilitate recycling of receptor complexes and/or allow the transduction of external stimuli into the cell.

Assessment and discrimination of odor stimuli in rat olfactory bulb by dynamic functional MRI

Dynamic blood oxygenation level-dependent functional MRI was applied at 7 T in the rat olfactory bulb (OB) with pulsed delivery of iso-amyl acetate (IAA) and limonene. Acquisition times for single-slice and whole OB data were 8 and 32 s, respectively, with spatial resolution of 220 × 220 × 250 μm. On an intrasubject basis, short IAA exposures of 0.6 min separated by 3.5-min intervals induced reproducible spatial activity patterns (SAPs) in the olfactory nerve layer, glomerular layer, and external plexiform layer. During long exposures (≈10 min), the initially dominant dorsal SAPs declined in intensity and area, whereas in some OB regions, the initially weak ventral/lateral SAPs increased first and then decreased. The SAPs of different concentrations were topologically similar, which implies that whereas an odor at various concentrations activates the same subsets of receptor cells, different concentrations are assessed and discriminated by variable magnitudes of laminarspecific activations. IAA and limonene reproducibly activated different subsets of receptor cells with some overlaps. Whereas qualitative topographical agreement was observed with results from other methods, the current dynamic blood oxygenation level-dependent functional MRI results can provide quantitative SAPs of the entire OB.