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Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires' disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing., (© 2021. The Author(s).)

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues., Competing Interests: The authors have declared that no competing interests exist.

Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis., Competing Interests: Conflict of interest disclosure The authors have no conflicts of interest to declare., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Among 98 serotypes of Streptococcus pneumoniae, only a small subset regularly causes invasive pneumococcal diseases (IPD). We previously demonstrated that serotype 11A binds to ficolin-2 and has low invasiveness in children. Epidemiologic data suggested, however, that serotype 11A IPD afflicts older adults, possibly indicating reduced ficolin-2-mediated immune protection. Therefore, we studied the epidemiology of ficolin-2-bound serotypes. We obtained IPD case data from the United States Centers for Disease Control and Prevention. We studied three prominent ficolin-2-bound serotypes and their acetyltransferase-deficient variants for ficolin-2 binding and ficolin-2-mediated complement deposition with flow-cytometry. We determined the age distributions of these serotypes from the obtained epidemiologic data. We discovered that the serotype 35B capsule is a novel ficolin-2 ligand due to O-acetylation via WciG. Ficolin-2-mediated complement deposition was observed on serotypes 11A and 35B but not serotype 31 or any O-acetyl transferase deficient derivatives of these serotypes. Serotypes 11A, 35B, and 31 cause more IPD among older adults than children. Studies of the three serotypes provide additional evidence for ficolin-2 providing innate immunity against IPD. The skewed age distribution of the three serotypes suggests that older adults have reduced ficolin-2-mediated immunity and are more susceptible to these serotypes., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: The University of Alabama at Birmingham (UAB) has intellectual property rights on some reagents developed in my laboratory, and all authors were UAB employees a the time these studies were performed. The authors have declared that no additional competing interests exist. This does note alter our adherence to PLOS ONE on policies on sharing data and materials.

Plants evolved disease resistance (R) proteins that recognize corresponding pathogen effectors and activate effector-triggered immunity (ETI). However, it is largely unknown why, in some cases, a suppressor of ETI exists in plants. Arabidopsis SOBER1 (Suppressor of AvrBsT-elicited Resistance 1) was identified previously as a suppressor of Xanthomonas acetyltransferase effector AvrBsT-triggered immunity. Nevertheless, the extent to which SOBER1 suppresses ETI is unclear. Here, we identified SOBER1 as a suppressor of Pseudomonas acetyltransferase effector HopZ5-triggered immunity in Arabidopsis using recombinant inbred lines. Further analysis showed that SOBER1 suppresses immunity triggered by multiple bacterial acetyltransferases. Interestingly, SOBER1 interferes with the immunity signalling activated by some but not all tested acetyltransferase effectors, indicating that SOBER1 might target components that are shared between several ETI pathways., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Viruses exploit the host and induce drastic metabolic changes to ensure an optimal environment for replication and the production of viral progenies. In response, the host has developed diverse countermeasures to sense and limit these alterations to combat viral infection. One such host mechanism is through interferon signaling. Interferons are cytokines that enhances the transcription of hundreds of interferon-stimulated genes (ISGs) whose products are key players in the innate immune response to viral infection. In addition to their direct targeting of viral components, interferons and ISGs exert profound effects on cellular metabolism. Recent studies have started to illuminate on the specific role of interferon in rewiring cellular metabolism to activate immune cells and limit viral infection. This review reflects on our current understanding of the complex networking that occurs between the virus and host at the interface of cellular metabolism, with a focus on the ISGs in particular, cholesterol-25-hydroxylase (CH25H), spermidine/spermine acetyltransferase 1 (SAT1), indoleamine-2,3-dioxygenase (IDO1) and sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1), which were recently discovered to modulate specific metabolic events and consequently deter viral infection., Competing Interests: The authors declare no conflict of interests.

Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S. aureus systemic reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S. aureus promotes robust T cell proliferation, but limits Th cells polarization and production of IL-1β and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S. aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S. aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1β, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S. aureus modulates cytokines critical for induction of protective Th17 immunity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Type-III secreted effectors (T3Es) play critical roles during bacterial pathogenesis in plants. Plant recognition of certain T3Es can trigger defence, often accompanied by macroscopic cell death, termed the hypersensitive response (HR). Economically important species of kiwifruit are susceptible to Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker. Although Psa is non-pathogenic in Arabidopsis thaliana, we observed that a T3E, HopZ5 that is unique to a global outbreak clade of Psa, triggers HR and defence in Arabidopsis accession Ct-1. Ws-2 and Col-0 accessions are unable to produce an HR in response to Pseudomonas-delivered HopZ5. While Ws-2 is susceptible to virulent bacterial strain Pseudomonas syringae pv. tomato DC3000 carrying HopZ5, Col-0 is resistant despite the lack of an HR. We show that HopZ5, like other members of the YopJ superfamily of acetyltransferases that it belongs to, autoacetylates lysine residues. Through comparisons to other family members, we identified an acetyltransferase catalytic activity and demonstrate its requirement for triggering defence in Arabidopsis and Nicotiana species. Collectively, data herein indicate that HopZ5 is a plasma membrane-localized acetyltransferase with autoacetylation activity required for avirulence.

Modifications of plant immune complexes by secreted pathogen effectors can trigger strong immune responses mediated by the action of nucleotide binding-leucine-rich repeat immune receptors. Although some strains of the pathogen Pseudomonas syringae harbor effectors that individually can trigger immunity, the plant's response may be suppressed by other virulence factors. This work reveals a robust strategy for immune suppression mediated by HopZ3, an effector in the YopJ family of acetyltransferases. The suppressing HopZ3 effector binds to and can acetylate multiple members of the RPM1 immune complex, as well as two P. syringae effectors that together activate the RPM1 complex. These acetylations modify serine, threonine, lysine, and/or histidine residues in the targets. Through HopZ3-mediated acetylation, it is possible that the whole effector-immune complex is inactivated, leading to increased growth of the pathogen., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1-resistance complex, resulting in ETI activation.

Recruitment of effector T cells to sites of infection or inflammation is essential for an effective adaptive immune response. The chemokine CCL5 (RANTES) activates its cognate receptor, CCR5, to initiate cellular functions, including chemotaxis. In earlier studies, we reported that CCL5-induced CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Specifically, CCL5-mediated mTOR activation contributes to T cell chemotaxis by initiating the synthesis of chemotaxis-related proteins. Up-regulation of chemotaxis-related proteins may prime T cells for efficient migration. It is now clear that mTOR is also a central regulator of nutrient sensing and glycolysis. Herein we describe a role for CCL5-mediated glucose uptake and ATP accumulation to meet the energy demands of chemotaxis in activated T cells. We provide evidence that CCL5 is able to induce glucose uptake in an mTOR-dependent manner. CCL5 treatment of ex vivo activated human CD3(+) T cells also induced the activation of the nutrient-sensing kinase AMPK and downstream substrates ACC-1, PFKFB-2, and GSK-3β. Using 2-deoxy-d-glucose, an inhibitor of glucose uptake, and compound C, an inhibitor of AMPK, experimental data are presented that demonstrate that CCL5-mediated T cell chemotaxis is dependent on glucose, as these inhibitors inhibit CCL5-mediated chemotaxis in a dose-dependent manner. Altogether, these findings suggest that both glycolysis and AMPK signaling are required for efficient T cell migration in response to CCL5. These studies extend the role of CCL5 mediated CCR5 signaling beyond lymphocyte chemotaxis and demonstrate a role for chemokines in promoting glucose uptake and ATP production to match energy demands of migration.

Aims/hypothesis: The aim of our study was to analyze combined impact of 17 polymorphisms at 8 gene regions previously shown to be associated with autoimmunity in diabetes. We hypothesized that the genetic predisposition is multiplicative and joint risk of different diabetic phenotypes forms by distinct combination of susceptibility loci., Methods: An ethnically homogenous population of Estonian origin, including 65 LADA patients, 154 patients with T1D, 260 patients with T2D and 229 non-diabetic controls, was genotyped for polymorphisms/haplotypes in HLA-DQB1, insulin gene (rs689, rs3842729), PHTF1-PTPN22 region (rs2476601, rs6679677), CTLA4 region (rs231806, rs16840252, rs5742909, rs231775, rs3087243, rs2033171), ICOS region (rs10932037, rs4675379), CD25 (rs706778), CD226(rs763361), NAA25 (rs17696736)., Results: As expected, the risk of T1D was consistently attributed by HLA-DQB1 haplotypes, but also by haplotypes of INS and PHTF1-PTPN22 region, and rs17696736 in NAA25. By contrast, LADA was associated only with T1D-protective HLA haplotypes and with two more frequent haplotypes of the CTLA4. It is of interest, that seldom CT haplotype of PHTF1-PTPN22 region carried the risk for autoantibody-negative T2D. The final best-fitted model for T1D genetic risk contained six gene regions (HLA-DQB1, INS, PHTF1, CTLA4 +49, CD226 and NAA25) and for LADA only two (HLA-DQB1 and CTLA4 +49). The AUCs of these models are 0.869 and 0.693, respectively., Conclusions: Classical T1D-risk haplotypes of HLA and some non-HLA loci describe quite well the genetic risk for T1D but not for LADA. The need of further studies should be stressed to discover the real risk factors for slower forms of autoimmune diabetes in adults., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Microbial pathogens have evolved mechanisms to overcome immune responses and successfully infect their host. Here, we studied how Listeria monocytogenes evades immune detection by peptidoglycan (PGN) modification. By analyzing L. monocytogenes muropeptides, we detected O-acetylated muramic acid residues. We identified an O-acetyltransferase gene, oatA, in the L. monocytogenes genome sequence. Comparison of PGN from parental and isogenic oatA mutant strains showed that the O-acetyltransferase OatA O-acetylates Listeria PGN. We also found that PGN O-acetylation confers resistance to different types of antimicrobial compounds targeting bacterial cell wall such as lysozyme, β-lactam antibiotics, and bacteriocins and that O-acetylation is required for Listeria growth in macrophages. Moreover, oatA mutant virulence is drastically affected in mice following intravenous or oral inoculation. In addition, the oatA mutant induced early secretion of proinflammatory cytokines and chemokines in vivo. These results suggest an important role for OatA in limiting innate immune responses and promoting bacterial survival in the infected host.

Approximately 84% of legionellosis cases are due to Legionella pneumophila serogroup 1. Moreover, a majority of L. pneumophila serogroup 1 clinical isolates react positively with monoclonal antibody 2 (MAb2) of the international standard panel. Over 94% of the legionellosis outbreaks investigated by the Centers for Disease Control and Prevention are due to this subset of L. pneumophila serogroup 1. To date, there is no complete explanation for the enhanced ability of these strains to cause disease. To better characterize these organisms, we subtyped 100 clinical L. pneumophila serogroup 1 isolates and 50 environmental L. pneumophila serogroup 1 isolates from the United States by (i) reactivity with MAb2, (ii) presence of a lag-1 gene required for the MAb2 epitope, and (iii) sequence-based typing analysis. Our results showed that the MAb2 epitope and lag-1 gene are overrepresented in clinical L. pneumophila serogroup 1 isolates. MAb2 recognized 75% of clinical isolates but only 6% of environmental isolates. Similarly, 75% of clinical isolates but only 8% of environmental isolates harbored lag-1. We identified three distinct lag-1 alleles, referred to as Philadelphia, Arizona, and Lens alleles, among 79 isolates carrying this gene. The Arizona allele is described for the first time in this study. We identified 59 different sequence types (STs), and 34 STs (58%) were unique to the United States. Our results support the hypothesis that a select group of STs may have an enhanced ability to cause legionellosis. Combining sequence typing and lag-1 analysis shows that STs tend to associate with a single lag-1 allele type, suggesting a hierarchy of virulence genotypes. Further analysis of ST and lag-1 profiles may identify genotypes of L. pneumophila serogroup 1 that warrant immediate intervention.

The HIV Tat-interacting protein (TIP30), also called CC3 or HTIP2, is encoded by Tip30, a putative tumor-suppressor gene located on human chromosome 11p15.1. In this study, we investigated the role of TIP30 in the progression and metastasis of lung cancer. TIP30 expression was analyzed in 206 paired lung cancers and adjacent non-tumor tissues, as well as in 70 matched lymph node metastases using a high-density tissue microarray. Results were compared with the clinicopathologic features of the patients from whom the tissues were taken. Low TIP30 expression levels were found in all 9 cases of small cell lung cancer and in 36.5% (72/197) of non-small cell lung cancer, which were correlated with lymph node metastasis in non-small cell lung cancer and with poor differentiation and advanced stage of tumor cells in squamous cell carcinoma. The immunostaining scores were significantly lower in the metastatic lesions than in the primary lesions. Down-regulation of TIP30 by a short hairpin RNA enhanced cell survival, migration, and invasion through Matrigel in vitro, and promoted lung metastasis and vascularization in nude mice. Further studies revealed that the down-regulation of TIP30 enhanced the expression of osteopontin, as well as matrix metalloproteinase-2 and vascular endothelial growth factor. Our results suggest that the down-regulation of TIP30 promotes metastatic progression of lung cancer, hence it could serve as a potential target for the development of lung cancer therapies.

An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE31-bar) and efficiently purified by Ni2+ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: 0.01 microg/ml) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [4.9 +/- 0.4 microg/g of sample (n = 6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor alpha (ERalpha) or activated ERbeta is present. Both the activation function 1 and the ligand binding domains of ERalpha are required for maximal induction. Mutations in the coactivator binding groove of the ERalpha ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERalpha must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERalpha mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERalpha DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERalpha and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.

We have obtained a specific monoclonal antibody (MAb) against the Plasmodium falciparum histone acetyl transferase (PfGcn5), a transcriptional factor that possesses HAT activity directed to the amino terminal of histone H3. To prepare this antibody, a 968-base pair (bp) DNA fragment of PfGcn5 gene corresponding to C-terminal domain was obtained by RT-PCR and cloned into prokaryotic expression vector pGEX-4T3. MAb against PfGcn5 was obtained with hybridoma technique and ELISA screening using either purified GSTPfGcn5 protein or purified GST protein alone as a control. One MAb, named Pf.r2, was able to identify the PfGcn5 protein in nuclear extract from P. falciparum and immunofluorescence assays. This MAb will be a helpful tool to perform a variety of assays to identify the other components of PfGcn5 complexes.

Prostate cancer (CaP) is initially androgen sensitive and responsive to hormone ablation therapy. However, cancer growth recurs despite androgen deprivation in the majority of cases of advanced disease. The molecular basis of this progression still remains unknown. The significance of androgen receptor (AR) coactivator proteins in this androgen-dependent malignancy is only beginning to emerge. In the present study, we examined the role of Tat interactive protein, 60 kDa (Tip60), an AR coactivator, in CaP progression. In hormone refractory CaP biopsies, we observed a nuclear accumulation of Tip60 expression in contrast to a more diffuse distribution pattern observed in benign prostate hyperplasia and primary CaP. Furthermore, in both the prostate xenograft model CWR22 and the LNCaP CaP cell line, we observed that androgen withdrawal promoted upregulation of Tip60 as well as nuclear accumulation. In contrast, androgen exposure resulted in decreased Tip60 expression that was more closely linked to a cytoplasmic presence. Chromatin immunoprecipitation analysis revealed Tip60's recruitment to the PSA gene promoter in both androgen-dependent and -independent cell lines. Thus, in vitro and in vivo data support a possible role for Tip60 in the molecular pathway leading to the development of androgen-independent CaP following long-term androgen deprivation therapy.

The Esa1-containing NuA4 histone acetylase complex can interact with activation domains in vitro and stimulate transcription on reconstituted chromatin templates. In yeast cells, Esa1 is targeted to a small subset of promoters in an activator-specific manner. Esa1 is specifically recruited to ribosomal protein (RP) promoters, and this recruitment appears to require binding by Rap1 or Abf1. Esa1 is important for RP transcription, and Esa1 recruitment to RP promoters correlates with coordinate regulation of RP genes in response to growth stimuli. However, following Esa1 depletion, H4 acetylation decreases dramatically at many loci, but transcription is not generally affected. Therefore, the transcription-associated targeted recruitment of Esa1 to RP promoters occurs in a background of more global nontargeted acetylation that is itself not required for transcription.

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.

Purified histone acetyltransferase B (HAT-B) from maize consists of two subunits, p50 and p45. Cloning of the cDNA and genomic DNA encoding the catalytic subunit p50 revealed a consensus motif reminiscent of other acetyltransferases. Internal peptide sequences and immunological studies identified p45 as a protein related to the Retinoblastoma associated protein Rbap. Antibodies against recombinant p50 were able to immunoprecipitate the enzymatic activity of p50 as well as p45. Consistent with the idea that HAT-B is involved in acetylation of newly synthesized histone H4 during DNA replication, mRNA and protein levels are correlated with S-phases during embryo germination. Inhibition of histone deacetylases by HC toxin or Trichostatin A caused a decrease of the in vivo expression of HAT-B mRNA. Regardless of its predominant cytoplasmic localization, a significant proportion of HAT-B-p50 is present in nuclei, irrespective of the cell cycle stage, suggesting an additional nuclear function.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.

Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.

A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity of this domain. Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infected with homologous or heterologous meningococci or other neisserial species. These results have important implications for the understanding of PDHC and the design of future outer membrane vesicle-based vaccines.

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N'-bis-(3-D-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immuno-affinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and K(m) values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.

Formate is the major source of C1 units in many species of the genus Clostridium. In this study we have cloned and characterized the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum. The genetic and transcriptional organizations of the genes and the high level of homology exhibited by the respective gene products to their Escherichia coli counterparts indicate strong evolutionary conservation of these enzymes.

Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme catalyses the freely reversible interchange of an acetyl group between N-acetylornithine and glutamate and has a specific activity of 22 mumol.min-1.mg-1 at 37 degrees C and pH 7.5. The Km values were determined for the substrates of the forward and reverse directions to be 1.0 mM for N-acetylornithine, 7.2 mM for glutamate, 1.5 mM for ornithine and 17.1 mM for N-acetylglutamate. The enzyme was localised to the mitochondrial matrix and was found to be a 57-kDa heterodimer consisting of subunits of 31 kDa and 26 kDa. Antibodies raised against the small subunit immunoprecipitated a single in vitro translation product of approximately 57 kDa suggesting that the subunits are processed from a single precursor protein. This is supported by N-terminal sequence analysis which shows that the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) with the N-terminus of ornithine acetyltransferase from Neisseria gonorrhoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identity (9 out of 20) with a sequence located in the middle of the 60-kDa N. gonorrhoeae enzyme. The N-terminal sequence of the small subunit has the potential to form an amphiphilic helix, further suggesting that the precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits.

Trifluoroacetylated (CF3CO-) proteins, elicited upon exposure of animals or humans to halothane, were recognized by anti-CF3CO antibody, monospecific for the hapten derivative N6-trifluoroacetyl-L-lysine. Anti-CF3CO antibodies cross-reacted with the dihydrolipoamide acetyltransferase (E2 subunit) of pyruvate dehydrogenase, indicating that epitopes on the E2 subunit of pyruvate dehydrogenase molecularly mimic those on CF3CO-proteins. Lipoic acid, the prosthetic group of the E2 subunit of pyruvate dehydrogenase was essential in this process, in that only the lipoylated form of the recombinantly expressed inner lipoyl domain of the human E2 subunit of pyruvate dehydrogenase, but not the unlipolyated form, was recognized by anti-CF3CO antibody. Furthermore, based on a high degree of structural relatedness, both CF3CO-Lys and (6RS)-lipoic acid, as well as the lipoylated peptide ETDK(lipoyl)ATIG specifically inhibited the recognition by anti-CF3CO antibody of the E2 subunit of pyruvate dehydrogenase, of trifluoroacetylated rabbit serum albumin and of human liver CF3CO-proteins. In sera of patients with halothane hepatitis, autoantibodies with properties identical to those of anti-CF3CO antibody were identified which could not discriminate between CF3CO-proteins and the E2 subunit of pyruvate dehydrogenase. These data suggest that the E2 subunit pyruvate of dehydrogenase is an autoantigen in halothane hepatitis and that molecular mimicry of CF3CO-proteins by the E2 subunit of pyruvate dehydrogenase is due to the similar structures of CF3CO-Lys and lipoic acid.

A superinduction of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT) accompanies the phenotype-specific cytotoxic response to a class of antitumor polyamine analogues in several important human solid tumor models. A highly specific antiserum against the human SSAT protein has been developed. Using this antiserum we demonstrate that polyamine analogue treatment in vitro or in vivo results in an induction of SSAT protein which is uniformly distributed throughout the cytoplasm of treated tumor cells. This new biochemical tool will be useful in the examination of the association of superinduced SSAT activity and cell type-specific cytotoxicity. Additionally, since clinical trials have begun on one of the SSAT-inducing polyamine analogues, this antiserum may be useful as a diagnostic tool in differentiating responsive and nonresponsive tumor cell populations in treated patients.

Upon induction of their nodulation genes, the root nodule-inducing Rhizobium bacteria produce lipo-oligosaccharide signal molecules. All lipo-oligosaccharides identified from Rhizobium leguminosarum bv. viciae carry an O-acetyl group at the C-6 position of the non-reducing terminal sugar, the presence of which is important for biological activity and host specificity. Previously we showed that a functional nodL gene product is required for the presence of this O-acetyl moiety. The production of polyclonal antibodies against isolated NodL protein, using a NodL-overproducing Escherichia coli strain is described. These antibodies were used (i) to elucidate the subcellular localization of the NodL protein, which appeared to be present in the cytosol, and (ii) for the purification of native NodL protein from E. coli. Here we provide biochemical proof that purified NodL protein has transacetylating activity in vitro with acetyl-CoA as the acetyl donor. NodL protein appeared to be able to acetylate various substrates, such as lipo-oligosaccharides, chitin fragments and N-acetylglucosamine. For chitinpentaose as the substrate we have shown, using mass spectrometry and NMR spectroscopy, that NodL protein substitutes one O-acetyl group at the C-6 position of the non-reducing terminal sugar.

Physarum vitronectin-like protein with a molecular mass of 70 kDa cross-reacts with anti-bovine vitronectin and promotes cell-spreading (Miyazaki, K. et al. 1992. Exp. Cell Res., 199: 106-110.). The amino-terminal sequence of Physarum vitronectin-like protein is, however, distinct from those of animal vitronectins but shows significant sequence homology with dihydrolipoamide acetyltransferase, a component of pyruvate dehydrogenase complex. We have investigated the structural relationships between Physarum vitronectin-like protein and dihydrolipoamide acetyltransferase by using both antibody and protein-chemical methods. The vitronectin-like protein reacted with both anti-bovine vitronectin IgG and anti-rat pyruvate dehydrogenase complex IgG, indicating that it shares common antigenic determinant(s) with rat pyruvate dehydrogenase complex. Furthermore, sequencing studies of peptides obtained by lysylendopeptidase digestion indicated that internal sequences of Physarum vitronectin-like protein show significant homology with dihydrolipoamide acetyltransferase, but do not show any homology with the primary structures of authentic vitronectins. Immunocytochemistry revealed that the protein is widely localized in cytoplasm and nuclei of Physarum polycephalum, but is not present in the central area of vacuoles. Our results indicate that Physarum vitronectin-like protein is a molecule structurally and immunologically related to dihydrolipoamide acetyltransferase but functionally similar to animal vitronectin, although its localization is unique.

mRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the lambda phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC--dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2)--were isolated, appearing at a frequency of 0.01% in the combinatorial immunoglobulin library. These Fab clones recognize human PDC-E2 with high affinity (Ka = 10(-7)-10(-9) M-1). Using both immunoblotting and ELISA, LC1-LC6 showed little cross-reactivity to any of the other autoantigens commonly recognized by PBC sera or to other antigens commonly recognized by PBC sera or to other antigens such as histone, calf thymus DNA, and bovine serum albumin. The Fab monoclonal antibodies show a typical anti-mitochondrial staining pattern in HEp-2 cells but react strongly with the luminal aspect of biliary epithelial cells of patients with PBC. Our results demonstrate that a recombinant combinatorial immunoglobulin library can be used to isolate high-affinity Fabs against a specific autoantigen. Such reagents will facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.

Seventy amikacin-resistant clinical isolates of gram-negative bacteria belonging to nine genera were examined by immunoblotting and by DNA-DNA hybridisation for the presence of ACC(6')1b enzyme, previously called AAC(6')-4, or its encoding gene aacA1b. The organisms mostly had resistance profiles compatible with AAC(6') production and were from South and North America, the Far East and Europe. Polyclonal (rabbit) anti-AAC(6')-1b antisera and an intragenic aacA1b (aacA4) probe derived from the multiresistance plasmid pAZ007 were used. The aacA1b gene was found to be widespread. Positive hybridisation, and immunologically cross-reactive proteins, were observed in 44% of the isolates examined. They were present most frequently (greater than or equal to 70%) in isolates of Klebsiella, Escherichia and Enterobacter spp., but less often (less than or equal to 25%) in Serratia, Citrobacter, Acinetobacter and Pseudomonas spp. The strains that reacted with the probe produced enzymes that varied in their apparent mol. wts between c. 24,000 and 26,000. The existence of multiple electrophoretic forms of amikacin-acetylating enzymes of the ACC(6')-1b type may be useful in epidemiological surveys of AAC(6')-mediated amikacin resistance.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in the serum. The major antigens recognized by the antibodies are the E2 components of the 2-oxo acid dehydrogenase complexes, all of which possess covalently attached lipoic acid cofactors. A bacterial etiology has been proposed for the disease, and patients' antibodies are known to recognize the E2 subunits (E2p) of both mammalian and bacterial pyruvate dehydrogenase complexes. Immunoblotting and ELISA inhibition techniques using extracts of Escherichia coli deletion strains, genetically restructured E2 polypeptides, and isolated lipoyl domains demonstrate that (i) the E2o subunit of the E. coli 2-oxoglutarate dehydrogenase complex is recognized by patients' antibodies; (ii) the main immunogenic region of E2p lies within the lipoly domains; (iii) the presence of a lipoly residue within the domain is crucial for effective recognition by the antibodies; and (iv) octanoylated E2p, octanoylated E2o, and octanoylated lipoyl domain, produced by a mutant deficient in lipoate biosynthesis, are recognized by patients' antibodies but not as effectively as their lipoylated counterparts. These findings indicate that antibodies in PBC patients' sera bind to a unique peptide-cofactor conformation within the lipoyl domains of the E2 polypeptides and that this epitope is partially mimicked by substituting the lipoyl cofactor with an octanoyl group.

Three plasmid-mediated chloramphenicol acetyltransferases isolated from different Haemophilus influenzae strains were purified and characterized. All three enzymes had properties in common with the gram-negative family of chloramphenicol acetyltransferase. The Haemophilus enzymes and the enteric type II enzyme were sensitive to 5,5'-dithiobis(2-nitrobenzoic acid), gave the same elution patterns from a highly substituted resin containing a bound chloramphenicol base, and had similar reactions to antisera. All four differed from each other in subunit molecular weight, enzyme activity, and partial protein digestion patterns. The data suggest that the three Haemophilus enzymes belong to the less common type II group and are related, but is not identical, to each other and to the enteric type II enzyme.

The dihydrolipoyl transacetylase component, which serves as the structural core of mammalian pyruvate dehydrogenase complexes, is acetylated when treated with either pyruvate or with acetyl-CoA in the presence of NADH. Besides the dihydrolipoyl transacetylase component, we have found that another protein, referred to as protein X, is rapidly acetylated at thiol residues. Protein X remains fully bound to the transacetylase core under conditions that remove the pyruvate dehydrogenase and dihydrolipoyl dehydrogenase components. Mapping of 125I-tryptic peptides indicated that the transacetylase subunits and protein X are structurally distinct; however, under the same mapping conditions, there is considerable similarity in the positions of acetylated peptides derived from these subunits. Affinity-purified rabbit immunoglobulin G prepared against the dihydrolipoyl transacetylase core reacted exclusively with the transacetylase and with both its tryptic-derived inner domain and outer lipolyl-bearing domain. Those results further indicate that protein X is not derived from the transacetylase subunit Affinity-purified mouse antibody to protein X reacted selectively with large tryptic polypeptides derived from protein X and did not react with the inner domain of the transacetylase. However, the anti-protein X antibody did react with the intact transacetylase subunit, the lipoyl-bearing domain of the transacetylase, and weakly with the transsuccinylase component of the alpha-ketoglutarate dehydrogenase complex. This cross-reactivity reflected specificity of a portion of the polyclonal antibodies for a related structural region in the transacetylase and protein X (possibly a similar lipoyl-bearing region). Furthermore, a major portion of that polyclonal antibody was shown to react exclusively with protein X. Thus, protein X subunits differ substantially from transacetylase subunits but the two components have a region of structural similarity. We estimate that there are about 5 mol of protein X per mol of the kidney pyruvate dehydrogenase complex. Under a variety of conditions that result in a wide range of levels of acetylation of sites in the complex, about 1 acetyl group is incorporated into protein X per 10 acetyl groups incorporated into the transacetylase subunits per mol of complex. That ratio is close to the ratio of protein X subunits of transacetylase subunits in the complex, indicating that there are efficient mechanisms for acylation and deacylation of protein X.

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues. Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex. Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross-linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations. The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes. Identification of these M2 mitochondrial autoantigens and detailed knowledge of their structure will allow important questions concerning this autoimmune disease to be addressed.

DNA-DNA hybridization and immunoblotting were used to assess the relatedness between the 6'-N-acetyltransferases of type 4 encoded by plasmid pAZ007 from a clinical isolate of Serratia marcescens and those encoded by NR79 and R5. The absence of detectable DNA-DNA homology and of immunological cross-reactivity suggests the existence of at least two distinct 6'-N-acetyltransferase type 4 genes that mediate amikacin resistance in gram-negative bacilli.

Autoantibodies to mitochondrial antigens are characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), but the precise antigenic determinants recognized by these antibodies have not been defined. Recently, our laboratory identified a 1,370-bp rat liver cDNA clone that coded for a polypeptide recognized specifically by sera from patients with PBC but not by sera from patients with other forms of liver disease. This recombinant protein was identified as the 74-kD M2 mitochondrial inner membrane autoantigen, now known to be dihydrolipoamide acetyltransferase. In the present study, we have identified a 603-bp fragment that codes for a polypeptide containing all of the autoreactivity of the original clone. In addition, based on hydrophobicity/hydrophilicity plots of the amino acid sequence of this polypeptide segment, several peptides were synthesized and tested for reactivity by an inhibition assay using sera from patients with PBC. One peptide, defined by the amino acids AEIETDKATIGFEVQEEGYL, absorbed serum reactivity to the protein product of the original clone. Of particular interest was the finding that this peptide contains the lipoic acid binding site KATIGF of the dihydrolipoamide acetyltransferase found in the inner mitochondrial membrane. Thus, it appears that for this autoantigen, the target of the autoantibodies corresponds to a functional site of the dihydrolipoamide acetyltransferase.