A Posttranscriptional Pathway of CD40 Ligand mRNA Stability Is Required for the Development of an Optimal Humoral Immune Response.

CD40 ligand (CD40L) mRNA stability is dependent on an activation-induced pathway that is mediated by the binding complexes containing the multifunctional RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1) to a 3' untranslated region of the transcript. To understand the relationship between regulated CD40L and the requirement for variegated expression during a T-dependent response, we engineered a mouse lacking the CD40L stability element (CD40LΔ5) and asked how this mutation altered multiple aspects of the humoral immunity. We found that CD40LΔ5 mice expressed CD40L at 60% wildtype levels, and lowered expression corresponded to significantly decreased levels of T-dependent Abs, loss of germinal center (GC) B cells and a disorganized GC structure. Gene expression analysis of B cells from CD40LΔ5 mice revealed that genes associated with cell cycle and DNA replication were significantly downregulated and genes linked to apoptosis upregulated. Importantly, somatic hypermutation was relatively unaffected although the number of cells expressing high-affinity Abs was greatly reduced. Additionally, a significant loss of plasmablasts and early memory B cell precursors as a percentage of total GL7+ B cells was observed, indicating that differentiation cues leading to the development of post-GC subsets was highly dependent on a threshold level of CD40L. Thus, regulated mRNA stability plays an integral role in the optimization of humoral immunity by allowing for a dynamic level of CD40L expression on CD4 T cells that results in the proliferation and differentiation of pre-GC and GC B cells into functional subsets.

CopR of Sulfolobus solfataricus represents a novel class of archaeal-specific copper-responsive activators of transcription.

In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)(-1). Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.

Drosophila RB proteins repress differentiation-specific genes via two different mechanisms.

The RB and E2F proteins play important roles in the regulation of cell division, cell death, and development by controlling the expression of genes involved in these processes. The mechanisms of repression by the retinoblastoma protein (pRB) have been extensively studied at cell cycle-regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here, we have taken advantage of the simplicity of the E2F/RB pathway in flies to inspect the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex, dREAM/MMB, in a cell type- and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle-regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells, both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional mechanism of repression by the Polycomb group (PcG) protein Enhancer of zeste [E(Z)], which is involved in silencing of these genes through the dimethylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).

Response to excess copper in the hyperthermophile Sulfolobus solfataricus strain 98/2.

Copper is an essential micronutrient, but toxic in excess. Sulfolobus solfataricus cells have the ability to adapt to fluctuations of copper levels in their external environment. To better understand the molecular mechanism behind the organismal response to copper, the expression of the cluster of genes copRTA, which encodes the copper-responsive transcriptional regulator CopR, the copper-binding protein CopT, and CopA, has been investigated and the whole operon has been shown to be cotranscribed at low levels from the copR promoter under all conditions, whereas increased transcription from the copTA promoter occurs in the presence of excess copper. Furthermore, the expression of the copper-transporting ATPase CopA over a 27-h interval has been monitored by quantitative real-time RT-PCR and compared to the pattern of cellular copper accumulation, as determined in a parallel analysis by Inductively Coupled Plasma Optical Emission spectrometry (ICP-OES). The results provide the basis for a model of the molecular mechanisms of copper homeostasis in Sulfolobus, which relies on copper efflux and sequestration.

Development of an immunoglobulin M capture-based enzyme-linked immunosorbent assay for diagnosis of acute infections with Bartonella henselae.

We describe the development of an immunoglobulin M-specific enzyme-linked immunosorbent assay for the detection of an early antibody response to Bartonella henselae, the causative agent of cat scratch disease, bacillary angiomatosis, and endocarditis. This assay discriminates between B. henselae-positive and -negative patient samples with sensitivity and specificity values of 100% and 97.1%, respectively.