μ-chain-deficient mice possess B-1 cells and produce IgG and IgE, but Not IgA, following systemic sensitization and inhalational challenge in a fungal asthma model

Allergic bronchopulmonary aspergillosis is often difficult to treat and results in morbidity associated with chronic airway changes. This study assessed the requirement for B cells and their products in the allergic pulmonary phenotype in a murine model of fungal allergic asthma that mimics allergic bronchopulmonary aspergillosis. C57BL/6 and μMT mice (assumed to lack peripheral B cells) were sensitized with Aspergillus fumigatus extract and challenged with two inhalation exposures of live conidia to induce airway disease. Airway hyperresponsiveness after methacholine challenge, peribronchovascular inflammation, goblet cell metaplasia, and fibrotic remodeling of the airways was similar between μMT mice and their wild-type counterparts (C57BL/6). Surprisingly, even in the absence of the μ-chain, these μMT mice produced IgE and IgG Abs, although the Abs induced did not have specificity for A. fumigatus Ags. In contrast, IgA was not detected in either the lavage fluid or serum of μMT mice that had been exposed to A. fumigatus. Our findings also reveal the existence of CD19(+)CD9(+)IgD(+) B-1 cells in the lungs of the μMT animals. These data show the μMT mice to have a developmental pathway independent of the canonical μ-chain route that allows for their survival upon antigenic challenge with A. fumigatus conidia, although this pathway does not seem to allow for the normal development of Ag-specific repertoires. Additionally, this study shows that IgA is not required for either clearance or containment of A. fumigatus in the murine lung, as fungal outgrowth was not observed in the μMT animals after multiple inhalation exposures to live conidia.     

The 3��-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3′-extrahelical nucleotide (3′-flap) binding pocket. When presented with 5′-flap substrates having a 3′-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3′-flap on substrates reduced K_m and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3′-flap, and the absence of a 3′-flap from a 5′-flap substrate was more detrimental to hFEN1 activity than removal of the 5′-flap or introduction of a hairpin into the 5′-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3′-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5′-phosphorylated product. Together the results indicate that the presence of a 3′-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA)⁶ two-way junction from which a 5′-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5′-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5′-flap structures are imprecisely cleaved by DNA2 in a replication protein A -dependent manner (7). Subsequent to the DNA2 cleavage, Rad27 (yeast homologue of FEN1) cleaves precisely to generate an intermediate suitable for ligation (2). The recent discovery that human DNA2 is predominantly located in mitochondria in various human cell lines (8, 9) suggests that hFEN1 is the paramount 5′-flap endonuclease in the nuclei of human cells. This observation potentially provides a plausible rationale for why deletion of RAD27 (yeast FEN1 homologue) is tolerated in Saccharomyces cerevisiae (10), whereas deletion of FEN1 in mammals is embryonically lethal (11). Recent models wherein mice carrying a mutation (E160D) in the FEN1 gene, which was shown in vitro to alter enzymatic properties (12), have demonstrated that FEN1 functional deficiency in mice (S129 and Black 6) increases the incidence of cancer, albeit different types presumably due to genetic background (13, 14). Thus, the function of mammalian FEN1 in vivo is vital to the prevention of genomic instability. In addition to its importance in the nucleus, hFEN1 has recently been detected in mitochondrial extracts (15, 16) and implicated in mitochondrial long patch base excision repair (15). Considering the pivotal roles of hFEN1 in DNA replication and repair, it is of interest to understand how hFEN1 and homologues achieve substrate and scissile phosphate selectivity in the absence of sequence information.Since its initial discovery as a nuclease that completes reconstituted Okazaki fragment maturation (17) and subsequent rediscovery as a 5′-flap-specific nuclease (DNaseIV) from bacteria (18), mouse (19), and HeLa cells (20), FEN1 proteins ranging from phage to human have been studied biochemically, computationally, and structurally (5, 6, 21). Biochemical characterizations of FEN1 proteins from various organisms have shown that this family of nucleases can perform phosphodiesterase activity on a wide variety of substrates; however, the efficiency of catalysis on various substrates differs among the species. For instance, phage FEN1s prefer pseudo-Y substrates (22, 23), whereas the archaeal and eukaryotic FEN1s prefer 5′-flap substrates (21, 24, 25), which have two dsDNA domains, one upstream and downstream of the site of cleavage, and a 5′-ssDNA protrusion (Fig. 1A). Primary sequence analysis indicates that FEN1 proteins share characteristic N-terminal (N) and Intermediate (I) “domains,” which harbor the highly conserved carboxylate residues that bind the requisite divalent metal ions (26–28). Structural studies of FEN1 nucleases from phage to humans (22, 29–36), have shown that the N and I domains comprise a single nuclease core domain consisting of a mixed, six- or seven-stranded β-sheet packed against an α-helical structure on both sides. The α-helices on either side of the β-sheet are “bridged” by a helical arch that spans the active site groove (supplemental Fig. S1). On one side of the β-sheet, the α-helical bundle (αb1) creates the floor of the active site and a DNA binding motif (helix-3-turn-helix) (32). Similarly, the opposite α-helical bundle (αb2) has also been observed to interact with DNA (35). Based on site-directed mutagenesis studies with T5 phage FEN1 (T5FEN1) (37) and hFEN1 (38, 39), and crystallographic studies of T4 phage FEN1 (T4FEN1) (22) and Archaeoglobus fulgidus FEN1 (aFEN1) (35) in complex with DNA, a general model for how FEN1 proteins recognize flap DNA has emerged. The helix-3-turn-helix motif is involved in downstream dsDNA binding, whereas the upstream dsDNA domain is bound by αb2. The helical arch is likely involved in 5′-flap binding (22).Open in a separate windowFIGURE 1.Secondary structure schematics of hFEN1 substrates. A, illustration of a general flap substrate created using a bimolecular approach whereby a template strand (T-strand), which partially folds into a hairpin, anneals with the duplex strand (d-strand). The T-strand hairpin creates the upstream dsDNA domain, whereas the d-strand base pairs with the T-strand to create the downstream dsDNA domain. The flap or any other structure is created by addition of nucleotides to the 5′-end of the d-strand. The interface between the upstream and downstream dsDNA domains may be viewed as a derivative of a two-way junction (74). Annealing of either the F(5), E, or G(15) d-strands with the T3F T-strand results in the formation of a (B) double flap substrate (Flap of 5-nt d-strand paired with a Template with a 3′-Flap, F(5)·T3F), C, exonuclease substrate with a 3′-extrahelical nucleotide (EXO d-strand paired with a Template with a 3′-Flap, E·T3F), and a D, fork-GEN substrate with a 3′-extrahelical nucleotide and a 15-nt ssDNA gap capped by a 23-nt hairpin structure (fork-Gap of 15-nt d-strand paired with a Template with a 3′-Flap, G(15)·T3F). E, annealing the F(5) d-strand with the T oligonucleotide creates a single flap (Flap of 5-nt d-strand paired with a Template, F(5)·T).Unlike phage FEN1s, studies of FEN1s from eubacterial (40), archaeal (21), and eukaryotic origins (41) have shown that the addition of a 3′-extrahelical nucleotide (3′-flap) to the upstream duplex of a 5′-flap substrate results in a rate enhancement and an increase in cleavage site specificity. Moreover, substrates possessing a 3′-flap, which mimic physiological “equilibrating flaps,” were cleaved exactly one nucleotide into the downstream duplex, thereby resulting in 5′-phosphorylated dsDNA product that was a suitable substrate for DNA ligase I (21, 41). As postulated by Kaiser et al. (21), the structure of an archaeal FEN1 in complex with dsDNA with a 3′-overhang showed that the protein contains a cleft adjacent to the upstream dsDNA binding site that binds the 3′-flap by means of van der Waals and hydrogen bonding interactions with the sugar moiety (35). Once the residues associated with 3′-flap binding were identified, sequence alignment analyses showed that the amino acid residues in the 3′-flap binding pocket are highly conserved from archaea to human. Furthermore, mutation of the conserved amino acid residues in the 3′-flap binding pocket of hFEN1 resulted in reduced affinity for and cleavage specificity on double flap substrates (42). Although the effects of the addition of a 3′-flap to substrates on hFEN1 catalysis are known qualitatively, a detailed understanding of the relationship between changes in catalytic parameters and rate enhancement by the presence of a 3′-flap is unknown. Here, we describe a detailed kinetic analysis of hFEN1 using four well characterized DNA substrates and show that the presence of a 3′-flap on a substrate not only contributes to substrate binding (42), but also increases multiple and single turnover rates of reaction in the presence of near physiological monovalent salt concentrations. We also demonstrate that, like T5FEN1, hFEN1 is rate-limited by product release, and thus multiple turnover rates at saturating concentrations of substrate are predominantly a reflection of product release and not catalysis as was previously concluded (39). Furthermore, this study provides insight into the mechanism of hFEN1 substrate recognition.     

Role of MAP1B in axonal retrograde transport of mitochondria

The MAPs (microtubule-associated proteins) MAP1B and tau are well known for binding to microtubules and stabilizing these structures. An additional role for MAPs has emerged recently where they appear to participate in the regulation of transport of cargos on the microtubules found in axons. In this role, tau has been associated with the regulation of anterograde axonal transport. We now report that MAP1B is associated with the regulation of retrograde axonal transport of mitochondria. This finding potentially provides precise control of axonal transport by MAPs at several levels: controlling the anterograde or retrograde direction of transport depending on the type of MAP involved, controlling the speed of transport and controlling the stability of the microtubule tracks upon which transport occurs.     

Involvement of Toll-like receptor 4 in acetaminophen hepatotoxicity

The objective of this study was to determine whether Toll-like receptor 4 (TLR4) has a role in alcohol-mediated acetaminophen (APAP) hepatotoxicity. TLR4 is involved in the inflammatory response to endotoxin. Others have found that ethanol-mediated liver disease is decreased in C3H/HeJ mice, which have a mutated TLR4 resulting in a decreased response to endotoxin compared with endotoxin-responsive mice. In the present study, short-term (1 wk) pretreatment with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, caused no histologically observed liver damage in either C3H/HeJ mice or endotoxin-responsive C3H/HeN mice, despite an increase in nitrotyrosine levels in the livers of C3H/HeN mice. In C3H/HeN mice pretreated with the alcohols, subsequent exposure to APAP caused a transient decrease in liver nitrotyrosine formation, possibly due to competitive interaction of peroxynitrite with APAP producing 3-nitroacetaminophen. Treatment with APAP alone resulted in steatosis in addition to congestion and necrosis in both C3H/HeN and C3H/HeJ mice, but the effects were more severe in endotoxin-responsive C3H/HeN mice. In alcohol-pretreated endotoxin-responsive C3H/HeN mice, subsequent exposure to APAP resulted in further increases in liver damage, including severe steatosis, associated with elevated plasma levels of TNF-alpha. In contrast, alcohol pretreatment of C3H/HeJ mice caused little to no increase in APAP hepatotoxicity and no increase in plasma TNF-alpha. Portal blood endotoxin levels were very low and were not detectably elevated by any of the treatments. In conclusion, this study implicates a role of TLR4 in APAP-mediated hepatotoxicity.     

Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids

The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 +/- 0.05 mM and a k(cat) of 60 s(-1) per active site (at 37 degrees C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 A resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.     

A ten-month study of endogenous testosterone levels and behaviour in outdoor-living female rhesus monkeys (Macaca mulatta)

Endogenous testosterone levels were measured in association with sexual, aggressive, and social/affiliative behaviors in 11 outdoor-housed female rhesus monkeys over a ten-month period. Several behaviors (sex directed toward the male, sex received from the male, aggression directed toward the male, submission directed toward the male, submission directed toward the female, and groom another female) were significantly (p<0.05) positively correlated with testosterone in from one to five females. No trends were strong enough across all females to suggest that any of these correlations have species-wide significance. Factor analysis revealed clearcut clusters of behaviors, but elevations in testosterone were not strongly associated with any of these clusters. It is concluded that endogenous testosterone levels have little measurable effect on overt behavior in female rhesus monkeys.