A Fluorescence-Based Assay for N5 -Carboxyaminoimidazole Ribonucleotide Mutase.

The enzyme N5 -carboxylaminoinidazole ribonucleotide (N5 -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N5 -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5 -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N5 -CAIR synthetase. During our research on identifying inhibitors of N5 -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N5 -CAIR mutase.

Window to His World: Using a Patient's YouTube Channel to Help Diagnose Chronic Mania.

Although chronic mania has been investigated, with several case reports and systematic retrospective cohort studies in the literature, it not a widely recognized entity. No specific definition for chronic mania is provided in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Furthermore, it is challenging to identify patients with chronic mania unless they come to the attention of the legal or medical system. We present the case of a manic patient who was hospitalized and subsequently found to have a YouTube channel that he had been using to promote his self-invented religion for over 2 years. Consent was obtained from the patient to review this YouTube channel for collateral information. From these videos, the patient was seen to be chronically circumstantial in his thought processes, grandiose in his ideas, highly energetic, distractible, preoccupied with religion, and talking with elaborate and rapid speech. A significant improvement in his symptoms was observed after administration of oral risperidone, with his scores on the Young Mania Rating Scale and Brief Psychiatric Rating Scale also showing improvement. To our knowledge, this is the first case in the literature in which an online video-sharing service was used longitudinally to facilitate diagnosis of a mental illness. We suggest that technology has great potential to improve our diagnostic tools, especially for disorders such as chronic mania the diagnosis of which relies primarily on self-report and collateral information.

Isatins Inhibit N5-CAIR Synthetase by a Substrate Depletion Mechanism.

The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthesize purines differently than humans. Microorganisms utilize two enzymes, N5-CAIR synthetase and N5-CAIR mutase, to convert 5-aminoimidazole ribonucleotide (AIR) into 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) through the intermediate N5-carboxy-5-aminoimidazole ribonucleotide (N5-CAIR). In contrast, vertebrates directly convert AIR to CAIR via the enzyme AIR carboxylase. A high-throughput screen against N5-CAIR synthetase identified a group of compounds with a 2,3-indolinedione (isatin) core that inhibited the enzyme. While initial studies suggested that isatins inhibited the enzyme by a noncompetitive mechanism, here we show that isatins inhibit N5-CAIR synthetase by a substrate depletion mechanism. Unexpectedly, we found that isatin reacts rapidly and reversibly with the substrate AIR. The rate of the reaction is dependent upon the substituents on the phenyl moiety of isatin, with 5- and 7-bromoisatin being faster than 4-bromoisatin. These studies suggest that care should be taken when exploring isatin compounds because the biological activity could be a result of their reactivity.

Functional analysis of the CpsA protein of Streptococcus agalactiae.

Streptococcal pathogens, such as the group B streptococcus (GBS) Streptococcus agalactiae, are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA-binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the ΔcpsA strain produced less capsule than did the wild type and demonstrated that the production of full-length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, the production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild-type background resulted in a dominant-negative decrease in capsule production. GBS expressing CpsA-245, but not the ΔcpsA strain, was attenuated in human whole blood. However, the ΔcpsA strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild-type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis but also cell wall associated factors.

C/EBPß mediates growth hormone-regulated expression of multiple target genes.

Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein ß (C/EBPß). This study examines the role of C/EBPß in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPß depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPß led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPß mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPß at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPß and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPß. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPß, and recruitment of p300. Overall, these studies suggest that C/EBPß, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH.

Restriction of rift valley Fever virus virulence in mosquito cells.

Arboviruses are maintained in a natural cycle that requires blood-sucking arthropod and vertebrate hosts. Arboviruses are believed to persistently infect their arthropod host without overt pathology and cause acute infection with viremia in their vertebrate host. We have focused on elucidating how a specific arbovirus, Rift Valley fever (RVF) virus, causes cytopathic effect in cells derived from vertebrates and non-cytopathic infection in cells derived from arthropods. We demonstrate that the vertebrate virulence factor, NSs, is functional in arthropod cells but is expressed at significantly lower levels in infected arthropod versus infected vertebrate cells.