Catalytic and DNA-binding properties of the human Ogg1 DNA N-glycosylase/AP lyase: biochemical exploration of H270, Q315 and F319, three amino acids of the 8-oxoguanine-binding pocket

The human Ogg1 protein (hOgg1) is an antimutator DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-7,8-dihydroguanine (8-oxoG) and the incision of apurinic and apyrimidinic (AP) sites in DNA. In this study, we have investigated the functional role of H270, Q315 and F319, three amino acids that are located in the 8-oxoG-binding pocket of hOgg1. Wild-type and mutant hOgg1 proteins (H270A, H270R, H270L, Q315A and F319A) were purified to apparent homogeneity. The catalytic activities and the DNA-binding properties of the various hOgg1 mutants were compared to those of the wild-type. The results show that hOgg1 mutated at H270 (H270A and H270L) or F319 (F319A) exhibits greatly reduced (50- to 1000-fold) DNA glycosylase activity, whereas the AP lyase activity is only moderately affected (<4-fold). The affinity of the hOgg1 mutants (H270A, H270L and F319A) for 8-oxoG.C-containing DNA is also greatly reduced (>30-fold), whereas their affinity for THF.C-containing DNA is only moderately reduced (<7-fold). The results also show that hOgg1 mutated at Q315 (Q315A) exhibits catalytic and DNA-binding properties similar to those of the wild-type. Therefore, H270 and F319 are essential to form the functional 8-oxoG-binding pocket, whereas Q315 is less crucial. In contrast, H270, Q315 and F319 are not required for efficient binding of THF.C and cleavage of AP sites. Finally, hOgg1 mutant proteins with a substitution of H270A or F319A are members of a new type of hOgg1 that is deficient in DNA glycosylase but proficient in AP lyase.     

Cellular and genetic analysis of wound healing in Drosophila larvae

To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution.     

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.     

Calcium measurement in living filamentous fungi expressing codon-optimized aequorin

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.     

Effects of antibody on viral kinetics in simian/human immunodeficiency virus infection: implications for vaccination

Passive antibody treatment of macaques prior to simian/human immunodeficiency virus infection produces "sterilizing immunity" in some animals and long-term reductions in viral loads in others. Analysis of viral kinetics suggests that antibody mediates sterilizing immunity by its effects on the initial viral inoculum. By contrast, reduction in peak viral load later in infection prevents CD4 depletion and contributes to long-term viral control.     

Signaling by the sea

In a spectacular location nestled in the hills on Croatia's coast, a group of 300 scientists from around the world gathered to listen to recent advances in cellular signaling and to gaze across the Adriatic Sea during discussions that surely put this work into perspective. Topics discussed ranged from precise structural details of signaling events to animal models for understanding signaling disorders.     

Adenylosuccinate lyase deficiency--first British case

A deficiency of adenylosuccinate lyase (ASDL) is characterised by the accumulation of SAICAriboside (SAICAr) and succinyladenosine (S-Ado) in body fluids. The severity of the clinical presentation correlates with a low S-Ado/SAICAr ratio in body fluids. We report the first British case of ADSL deficiency. The patient presented at 14 days with a progressive neonatal encephalopathy and seizures. There was marked axial and peripheral hypotonia. Brain MRI showed widespread white matter changes. She died at 4 weeks of age. Concentrations of SAICAr and SAdo were markedly elevated in urine, plasma and CSF and the SAdo/SAICAr ratio was low, consistent with the severe phenotype. The patient was compound heterozygous for 2 novel ADSL mutations; c.9 G>C (A3P) and c.572 C>T (R190X).     

Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.     

Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.Superinfection exclusion or homologous interference is a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, whereas infection by unrelated viruses can be unaffected. The phenomenon was first observed by McKinney (57, 58) between two genotypes of Tobacco mosaic virus (TMV) and later with bacteriophages (21, 94). Since that time, the phenomenon has been observed often for viruses of animals (1, 13, 18, 34, 43, 47, 50, 85, 86-88, 102, 103) and plants (11, 30, 31, 32, 39, 40, 49, 77, 99, 100). In plant virology, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses (58, 77). Subsequently, it was developed into a management tool to reduce crop losses by purposely infecting plants with mild isolates of a virus to reduce infection and losses due to more severe isolates, which is referred to as “cross-protection” (reviewed in references 32 and 40).Homologous superinfection exclusion of animal viruses has been related to several mechanisms acting at various stages of the viral life cycle, including prevention of the incoming virus entry into cells (50, 86, 87), or inhibition of translation or interference with replication (1, 47, 50, 83). Several mechanisms have been postulated for homologous interference of plant viruses, including prevention of the disassembly of the challenge virus as it enters the cell resulting from the expression of the coat protein of the protector virus (67, 84; reviewed in reference 10) and induction of RNA silencing by the protector virus that leads to sequence-specific degradation of the challenge virus RNA (24, 69, 70). However, common mechanisms of superinfection exclusion, expected to be associated with the viruses of plants and animals, have not been elucidated.Citrus tristeza virus (CTV) is the largest and most complex member of the Closteroviridae family, which contains viruses with mono-, bi-, and tripartite genomes transmitted by a range of insect vectors, including aphids, whiteflies, and mealybugs (3, 6, 19, 20, 46). CTV has long flexuous virions (2,000 nm by 10 to 12 nm) encapsidated by two coat proteins and a single-stranded RNA genome of ∼19.3 kb. The major coat protein (CP) covers ca. 97% of the genomic RNA, and the minor coat protein (CPm) completes encapsidation of the genome at its 5′ end (25, 81). The RNA genome of CTV encodes 12 open reading frames (ORFs) (44, 64) (Fig. ​(Fig.1).1). ORFs 1a and 1b are expressed from the genomic RNA and encode polyproteins required for virus replication. ORF 1a encodes a 349-kDa polyprotein containing two papainlike protease domains plus methyltransferaselike and helicaselike domains. Translation of the polyprotein is thought to occasionally continue through the polymerase-like domain (ORF 1b) by a +1 frameshift. Ten 3′-end ORFs are expressed by 3′-coterminal subgenomic RNAs (sgRNAs) (37, 45) and encode the following proteins: major (CP) and minor (CPm) coat proteins, p65 (HSP70 homolog), and p61 that are involved in assembly of virions (79); a hydrophobic p6 protein with a proposed role in virus movement (20, 89); p20 and p23, which along with CP are suppressors of RNA silencing (54); and p33, p13, and p18, whose functions remain unknown. Remarkably, citrus trees can be infected with mutants with three genes deleted: p33, p18, and p13 (89).Open in a separate windowFIG. 1.(A) Schematic diagram of the genome organization of wild-type CTV (CTV9R) and its derivative CTV-BC5/GFP encoding GFP. The open boxes represent ORFs and their translation products. PRO, papainlike protease domain; MT, methyltransferase; HEL, helicase; RdRp, an RNA-dependent RNA polymerase; HSP70h, HSP70 homolog; CPm, minor coat protein; CP, major coat protein; GFP, green fluorescent protein. Bent arrows indicate positions of BYV (B_CP) or CTV CP (C_CP) sgRNA controller elements. Inserted elements are shown in gray. (B) Scheme of the “superinfection exclusion assay.” Young Madam Vinous sweet orange trees were initially inoculated with one of 13 tested CTV isolates. When primary infections were established, the trees were subsequently challenged with CTV-BC5/GFP. All inoculations were done by grafting of the infected tissue into the stem of a tree. The positions of primary (Pri) and challenge (Chl) graft inoculations are shown. The ability of the challenge virus to superinfect trees was determined by visual observation of GFP fluorescence in phloem-associated cells on the internal surface of bark from a young flash starting at about 2 months upon challenge inoculation. Scale bar, 0.4 mm.The host range of CTV is limited to citrus in which the virus infects only phloem-associated cells. CTV consists of numerous isolates that have distinctive biological and genetic characteristics (38, 48, 56, 72, 74, 75, 95). Recently, a classification strategy for CTV isolates was proposed based on sequence similarity. Analysis of nearly 400 isolates in an international collection revealed five major CTV genotype groups with some isolates undefined (38). For the purposes of the present study, strains are defined as phylogenetically distinct lineages of CTV based upon analysis of nucleotide sequences of the 1a ORF (38). This region of the genome shows high genetic diversity between CTV variants, with levels of sequence identity ranging between 72.3 to 90.3% (38, 48, 52, 74, 75; M. Hilf, unpublished data). Using this definition, T3, T30, T36, VT, and T68 are designated as strains. Individual virus samples are designated as isolates of one of these strains. The ORF 1a nucleotide sequences of isolates of the T36 and T68 strains are equally dissimilar to isolates of the T3, T30, and VT strains, with identities of 72.9, 73, and 72.4% and 77.6, 77.9, and 76.8%, respectively. Identities of ORF 1a range from 89.4 to 90.3% between isolates of the T3, T30, and VT strains. Sequences of ORF1a of isolates belonging to the T36 strain and those from the T68 strain show 72.3% identity. This compares to a range of 89 to 94.8% identity found in the more conserved 3′-half regions of the genomes of isolates from different CTV strains. Each strain is named after a “type isolate” and is composed of isolates with minor sequence divergence (generally less than 5% throughout genome) from the type member. However, isolates of a strain may have significant variations in symptoms and symptoms severity. Remarkably, field trees harbor complex populations of CTV, which are often composed of mixtures of different strains and recombinants between these strains (36, 48, 52, 68, 75, 96, 101). The genetic basis of such frequent coexistence of different strains within the same tree is unknown.CTV causes economically important diseases of citrus worldwide. One of the most effective management tools has been cross-protection when effective protecting isolates could be found. Preinfection with mild isolates allows commercial production of sweet oranges and limes in Brazil (16) and Peru (9) and grapefruit in South Africa (92). However, identification of protecting isolates has been empirical, difficult, and rare. Cross-protection usually has worked only in certain varieties, and the lack of effective protecting isolates has prevented its use in many varieties and citrus growing areas (15, 41, 61, 73). In general, there has been no understanding why some mild isolates were effective and others failed to protect. Because CTV diseases prevail in citrus growing areas worldwide, elucidation of the mechanisms of exclusion of one CTV variant by another one is an important goal.In the present study we examined relationships between different genotypes of CTV in terms of their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between minor genetic variants of the same strain (sequence group) and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided full exclusion of the challenge isolate. In all combinations of virus isolates belonging to different strains, the primary infection of plants with one strain had no noticeable effect on the establishment of the secondary infection. The results obtained here help elucidate some previously unexplained fundamental features of CTV biology and pose the possibility of an existence of a novel mechanism for superinfection exclusion between virus variants.