Expanding the mass range for UVPD-based native top-down mass spectrometry.

Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles.

Reconstitution of CRISPR adaptation in vitro and its detection by PCR.

CRISPR adaptation is the initial step in CRISPR-Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1-Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Structural and biochemical studies from reconstituted complexes have provided mechanistic insight into how CRISPR adaptation occurs; however, these studies have been limited to a narrow subset of CRISPR-Cas types and may not be representative of the other types. Here we describe methods for the purification of the type I-F CRISPR adaptation complex (Cas1:Cas2-3) from Pectobacterium atrosepticum, purification of the DNA architectural protein integration host factor (IHF), and a sensitive PCR-based in vitro integration assay. This assay could easily be used to investigate mechanisms of CRISPR adaptation in other CRISPR-Cas systems, including the roles of accessory proteins.

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14-Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

Use of Saccharomyces cerevisiae in the identification of novel transcription factor DNA binding specificities.

Members of the steroid/hormone nuclear receptor superfamily regulate target gene transcription via recognition and association with specific cis-acting sequences of DNA, called hormone response elements (HREs). The identification of novel HREs is fundamental to understanding the physiological function of nuclear receptor-mediated signalling pathways. A number of these receptors are transcriptionally active, or can be induced to an active state, when expressed in the yeast strain Saccharomyces cerevisiae. This aspect of nuclear receptor activity was used to screen random rat genomic DNA fragments for their ability to function as a HRE for the farnesoid X-activated receptor (FXR). An isolated genomic fragment mediated FXR transcriptional activation without the co-expression of the retinoid-X receptor (RXR), a receptor previously thought to be an obligate heterodimer partner for FXR function. This genomic sequence of DNA contained a pair of highly conserved HRE half-sites arranged in an everted orientation and separated by 3 bp (ER3). Furthermore, it was located 240 bp from a highly conserved TATA box motif. A minimal ER3 sequence of DNA was further demonstrated to function as a FXR HRE and was bound in vitro by FXR-expressing yeast extracts. Using RT-PCR, an expressed mRNA fragment was identified within an 8 kb region downstream of the putative TATA box motif. This sequence of DNA was observed to bear homology to a cDNA found in mouse blastocyst. These findings define a novel FXR DNA binding specificity but, more importantly, these data suggest that this strategy might be universally applied to any transcription system that can be reconstituted in yeast.

Catabolites of cholesterol synthesis pathways and forskolin as activators of the farnesoid X-activated nuclear receptor.

The nuclear receptors are a family of transcriptional mediators that, upon activation, bind DNA and regulate gene transcription. Among these receptors, the farnesoid X-activated receptor (FXR) has recently been identified as one activated by bile acids and farnesol. To investigate the potential of other sterols to activate FXR, as well as to examine relevant relationships among identified activators of FXR, the current study used a mammalian cell transcription assay to quantify and compare activation potential. In addition to the classical bile acids deoxycholate (DCA) and chenodeoxycholate (CDCA), FXR was shown to be transcriptionally active in the presence of the androgen catabolites 5alpha-androstan-3alpha-ol-17-one (androsterone) and 5beta-androstan-3alpha-ol-17-one (etiocholanolone), as well as the sterol bronchodilatory drug forskolin. Conversely, cholesterol and several other key precursors to the androgens and bile acids were either not active or only slightly active. Furthermore, it was observed that the bile acid ursodeoxycholate (UDCA) could inhibit DCA and CDCA activation of FXR in a manner parallel to its ability to antagonize DCA and CDCA induction of apoptosis. By far, the most efficacious activator of FXR was forskolin. Interestingly, although it is classically viewed as an initiator of the adenylate cyclase/protein kinase A (PKA) pathway, PKA inhibition did not inhibit forskolin's activation of FXR nor was cyclic AMP (cAMP) able to stimulate FXR-mediated transcription. These data would suggest that forskolin acts as a ligand for FXR rather than as a secondary activator of FXR and could have important implications with respect to its potential toxicity and pharmacological use.

Assessment of the potential in vivo genotoxicity of three trihalomethanes: chlorodibromomethane, bromodichloromethane and bromoform.

Chlorination of drinking water results in the formation of chlorodibromomethane, bromodichloromethane and bromoform. These trihalomethanes have all shown evidence of genotoxicity in bacterial and mammalian cell systems in vitro and some evidence of carcinogenicity in rodents. Chlorodibromomethane and bromodichloromethane have previously been tested in the mouse micronucleus test and did not induce chromosome damage, but results from two previous micronucleus tests on bromoform are somewhat contradictory. In the present study, bromoform was tested in the mouse bone marrow micronucleus test in order to reassess the response in this system; all three compounds were evaluated using the rat liver unscheduled DNA synthesis test. Trihalomethanes are well absorbed by the oral route which was selected for this study as being that most relevant to humans. Bromoform did not induce micronuclei in mouse bone marrow, and chlorodibromomethane, bromodichloromethane and bromoform did not cause unscheduled DNA synthesis in rat liver. These trihalomethanes have not shown any evidence of genotoxicity in vivo and are most unlikely to have any significant genotoxic activity in mammals. Their mode of action as rodent carcinogens remains unexplained.

Nitrous oxide in the dental environment: assessing the risk, reducing the exposure.

This article identifies the potential sources of nitrous oxide in the dental environment and reviews the harmful effects of chronic exposure to nitrous oxide. The author suggests methods of controlling nitrous oxide concentrations to approximate the recommended levels in the dental care environment.

Assessment of the potential in vivo genotoxicity of fluoranthene.

Fluoranthene is a ubiquitous environmental pollutant. Although fluoranthene is mutagenic in bacterial and mammalian in vitro cell systems following metabolic activation by rat liver fraction, information on in vivo mutagenicity is lacking and studies on tumour initiating activity in mice are equivocal. In the present study, the potential genetic hazard to man was assessed using the mouse bone marrow micronucleus and rat liver unscheduled DNA synthesis test systems. Fluoranthene did not show any evidence of genotoxicity in either of the in vivo assays following acute oral administration at levels of up to 2000 mg/kg b.w.

Enhanced effectiveness of copper ion buffering by CUP1 metallothionein compared with CRS5 metallothionein in Saccharomyces cerevisiae.

The bakers' yeast Saccharomyces cerevisiae contains a metallothionein (MT) gene family comprised of the amplified CUP1 locus and the single copy CRS5 gene. We demonstrate that CUP1 plays the dominant role in copper detoxification. A single copy of CUP1 was far more effective in conferring copper resistance than was CRS5. The CUP1 promoter contributes to this resistance; in a promoter exchange experiment, the Crs5 MT conferred strong copper resistance when its expression was driven by the CUP1 promoter, and conversely, the CRS5 promoter reduced the effectiveness of Cup1 MT. Unlike CUP1, the CRS5 promoter appears to be refractory to high concentrations of copper. The CUP1 coding sequences also contribute to copper tolerance, presumably reflecting the enhanced binding avidity of Cup1 MT for Cu(I) ions. In studies with the bathocuproine Cu(I) chelator, the Cu(I) ions bound to Crs5 were kinetically more labile than the Cu(I) binding to Cup1. Our findings are consistent with the assembly of Crs5 into two metal-binding clusters, similar to mammalian MTs, but unlike Cup1. Overall, the striking differences in gene structure, regulation, and function of CUP1 and CRS5 are remarkably reminiscent of the MTI and MTII genes of the pathogenic yeast Candida glabrata.

CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae.

Protection from copper toxicity in the bakers' yeast Saccharomyces cerevisiae involves the action of a copper binding metallothionein encoded by the CUP1 locus. To identify additional factors contributing to copper ion homeostasis and detoxification, we screened a genomic library for genes that confer high levels of copper resistance to yeast strains lacking CUP1. This screen led to the identification of the CRS5 (copper-resistant suppressor) gene. By sequence analyses, CRS5 encodes a small molecular weight cysteine-rich protein with an amino acid sequence bearing all the features of a eukaryotic metallothionein. The CRS5 polypeptide exhibits a striking similarity to a number of mammalian and invertebrate metallothioneins, yet shares surprisingly little homology with CUP1. In yeast, CRS5 is expressed as a 0.5-kilobase mRNA that is regulated both by copper ions and by oxidative stress, and expression is dependent upon ACE1, a copper and DNA binding transcription factor also known to regulate CUP1. Deletion of the chromosomal CRS5 locus was found to increase cellular sensitivity to copper, but not cadmium, toxicity. These studies support an important role for the CRS5 metallothionein-like protein in copper homeostasis and detoxification.

Effect of different oxidation states of chromium in causing chromosome alterations in cultured CHO cells.

Four chromium salts with different oxidation states were tested for their influence in causing chromosome aberrations and sister-chromatid exchange in Chinese hamster ovary cells in vitro. Cell cultures were treated with CrO3, K2Cr2O7, CrCl2 and Cr(NO3)3.9H2O at concentrations of 10(-7), 10(-6), 10(-5) and 10(-4) M for the aberration assay, and 10(-8), 10(-7), 10(-6) and 10(-5) M for the sister-chromatid exchange assay. It was noticed that Cr (VI) compounds--CrO3 and K2Cr2O7--considerably enhanced the frequencies of aberrations and sister-chromatid exchanges compared to the control cultures. CrCl2 and Cr(NO3)3.9H2O--Cr (II) and Cr (III) respectively--caused a slight increase in sister-chromatid exchange rates, but the frequencies of aberrations were almost unchanged compared to the controls. These investigations indicate a definite link between the metals and changes produced in the mammalian chromosomes, reaffirming the evidence of carcinogenic potential of Cr (VI) observed by other investigators.

Patient-applied tooth whiteners.

A double-blind clinical trial compared the efficacy and safety of two popular carbamide peroxide tooth whitening products with a placebo agent. After six weeks, both study products produced significantly whiter teeth than did the placebo agent.

Verrucous carcinoma of the skin and its management by Mohs' surgery.

Verrucous carcinoma is a low-grade squamous cell cancer that can invade many anatomic sites. Since its clinical appearance is easily confused with several benign conditions, reliable diagnosis is by biopsy and histopathologic examination. The Mohs' histographic surgical technique provides complete surgical excision and a high incidence of cure.

Lymphatic complications of manual herpes simplex infection.

In seven patients with typical recurrent herpes simplex of the hand, lymphatic complications, developed in the arm during periods of herpetic activity. Six of the patients presented with lymphangitis and lymphadenitis. In one patient, persistent lymphedema developed in the involved hand and forearm.