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Rare or opportunistic fungal infections are mostly described in immunosuppressed patients. We present a case of a cutaneous phaeohyphomycosis that developed on the dorsal foot in an immunosuppressed woman suffering from AIDS, caused by a novel Phialemoniopsis species. It clinically presented as an indurated violaceous plaque, surmounted by nodules exuding a sero-purulent discharge. A filamentous fungus was isolated from pus and cutaneous biopsy. ITS and LSU sequences phylogenetically resolved the fungus as an unknown species of Phialemoniopsis, which is an unresolved family within Sordariomycetes. In this study we describe the new species as Phialemoniopsis limonesiae, which clusters on a single branch clearly separated from its closest phylogenetic neighbours. This new strain showed low MIC to itraconazole, voriconazole and posaconazole.

AIMS The purpose of this study was to investigate the regulatory mechanism of e-PL on Shewanella putrefaciens. METHODS AND RESULTS Proteomics analysis of inhibitory effect of e-PL against S. putrefaciens was performed by label-free quantitative assay based on high-resolution mass spectrometry (MS). Quantification of 2206 proteins was obtained with high confidence, and a total of 36 differentially expressed proteins (DEPs), with 10 and 26 proteins showing upregulation and downregulation, respectively, were identified. Upon Go functional enrichment, 11, 5 and 8 specific Go terms in biological processes, molecular functions and cellular components were identified, respectively. Six KEGG pathways, including 'ribosome', were significantly enriched. Among the ribosome pathway, there were seven DEPs and all of them were distributed on large and small subunits of ribosome. CONCLUSIONS The significant downregulation of proteins, large subunits of ribosomal proteins RP-L18, L30 and L27, small subunits ribosomal proteins S16 and S20, and RNA polymerase β' subunit protein rpoC were the critical action sites of e-PL to inhibit S. putrefaciens growth. SIGNIFICANCE AND IMPACT OF THE STUDY Shewanella putrefaciens is one of the representative fish-spoilage bacteria regardless of fish type, and poses significant problems for the fish brewery. A better understanding of the antibacterial mechanism of e-PL on S. putrefaciens could make important contributions to development of biological control strategies of these economically important pathogens.

SummaryPancreatic ductal adenocarcinoma (PDA) is one of the deadliest solid malignancies, with a 5-year survival rate at ten percent. PDA have unique metabolic adaptations in response to cell-intrinsic and environmental stressors, and identifying new strategies to target these adaptions is an area of active research. We previously described a dependency on a cytosolic aspartate aminotransaminase (GOT1)-dependent pathway for NADPH generation. Here, we sought to identify metabolic dependencies induced by GOT1 inhibition that could be exploited to selectively kill PDA. Using pharmacological methods, we identified cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. Targeting any of these pathways was synthetic lethal in GOT1 knockdown cells and triggered ferroptosis, an oxidative, non-apoptotic, iron-dependent form of cell death. Mechanistically, GOT1 inhibition promoted the activation of autophagy in response to metabolic stress. This enhanced the availability of labile iron through ferritinophagy, the autolysosome-mediated degradation of ferritin. In sum, our study identifies a novel biochemical connection between GOT1, iron regulation, and ferroptosis, and suggests the rewired malate-aspartate shuttle plays a role in protecting PDA from severe oxidative challenge.HighlightsPDA exhibit varying dependence on GOT1 forin vitroandin vivogrowth.Exogenous cystine, glutathione synthesis, and lipid antioxidant fidelity are essential under GOT1 suppression.GOT1 inhibition sensitizes pancreatic cancer cell lines to ferroptosis.GOT1 inhibition represses anabolic metabolism and promotes the release of iron through autophagy.

In May of 2008, a phytoplasma-like disease was observed on willows (Salix babylonica Linn) grown in the Shaanxi Province. Affected plants showed yellowed leaves with green veins and dieback. Incidence of the disease was less than 10%. Samples were collected from 10 symptomatic and five asymptomatic willow plants from five different areas in Shaanxi Province. Total DNA was extracted from 0.5 g of leaf midrib and stem phloem tissue with a modified cetyltrimethylammoniumbromide (CTAB) method (3). Resulting DNA extracts were analyzed by a nested PCR assay using phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 (1), which amplified a 1,452- and a 1,246-bp product, respectively. Sequences of amplicons were almost the same. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, HinfI, and TaqI endonucleases (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup C (16SrI-C) ‘Candidatus Phytoplasma asteris’. None of the symptomless plants tested positive. Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession No. FJ179166) confirmed the results on the basis of RFLP analyses. Subsequently, the presence of the phytoplasmas in symptomatic plants was also confirmed by transmission electron microscopy. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with a yellows-type disease of willows in northern China and its association with aster yellow group 16SrI, subgroup 16SrI-C. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Inst. J. Syst. Bacteriol. 48:1153, 1998. (3) Y. Qi et al. Biotechnol. Bull. 4:44, 2004.

Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5′-deoxyribose phosphate (5′-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro. The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro. The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.

The effects of chilling (CT, day/night temperatures of 12/10 °C, an irradiance of 250 μmol m−2 s−1), chilling combined with a low irradiance (CL, 12/10 °C, 80 μmol m−2 s−1), and a high temperature (HT, 42/40 °C, 250 μmol m−2 s−1) on chlorophyll content, chlorophyll fluorescence, and gas exchange were studied in two watermelon cultivars, ZJ8424 and YS01, differing in their resistance. The chlorophyll content, net photosynthetic rate (PN), stomatal conductance (gs), and transpiration rate (E) decreased substantially, whereas the intercellular CO2 concentration (ci) increased when the two watermelon cultivars were grown under these stresses. The photosynthetic parameters showed greater changes at chilling than at the high temperature, and the CL caused a more pronounced inhibition in PN compared with the CT. After 2 d exposure to the CT, YS01 had higher PN, gs, and E, but a lower ci compared with ZJ8424. The maximum efficiency of photosystem (PS) II photochemistry (Fv/Fm), effective quantum yield of PS II photochemistry (ΦPSII), photochemical quenching (qP), and electron transport rate (ETR) decreased under the CT and CL but showed only a slight drop under the HT. All these stresses significantly increased non-photochemical quenching (NPQ). The CT brought more damage to the photosynthetic apparatus of leaves compared with the CL. In addition, after returning to normal conditions (25/15 °C, 250 μmol m−2 s−1) for 3 d, the photosynthetic parameters recovered to pre-stress levels in HT treated seedlings but not in CT treated seedlings. In conclusion, the low irradiance could help to alleviate the extent of photoinhibition of PS II photochemistry caused by chilling and cv. ZJ8424 was more sensitive to the extreme temperatures than cv. YS01.

Objective: To review the safety and efficacy of obiltoxaximab, a monoclonal antibody indicated for the treatment of Bacillus anthracis inhalational anthrax in adult and pediatric patients. Data Sources: A MEDLINE (1946 to May, week 1, 2017) and EMBASE (1980 to 2017, week 19) search was performed using the search terms obiltoxaximab OR ETI-204 OR Anthim AND anthrax. Study Selection and Data Extraction: All English-language clinical studies in both animal and human models assessing the safety and efficacy of obiltoxaximab were included. Data Synthesis: A total of 5 articles have been published on clinical studies examining safety and efficacy of obiltoxaximab. Efficacy studies in 2 animal models, New Zealand White rabbits and cynomolgus macaques, showed higher rates of survival post–anthrax exposure when obiltoxaximab was administered. Safety studies in healthy human volunteers showed that it was tolerated, with a relatively low incidence of adverse events. Conclusion: Based on these clinical studies and the implausibility of conducting a trial in infected individuals, obiltoxaximab is a safe and efficacious addition to the anthrax antitoxin armamentarium to protect against and treat inhalational anthrax.

Objectives Several genome-wide association studies (GWAS) of bone mineral density (BMD) have successfully identified multiple susceptibility genes, yet isolated susceptibility genes are often difficult to interpret biologically. The aim of this study was to unravel the genetic background of BMD at pathway level, by integrating BMD GWAS data with genome-wide expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (meQTLs) data Method We employed the GWAS datasets of BMD from the Genetic Factors for Osteoporosis Consortium (GEFOS), analysing patients’ BMD. The areas studied included 32 735 femoral necks, 28 498 lumbar spines, and 8143 forearms. Genome-wide eQTLs (containing 923 021 eQTLs) and meQTLs (containing 683 152 unique methylation sites with local meQTLs) data sets were collected from recently published studies. Gene scores were first calculated by summary data-based Mendelian randomisation (SMR) software and meQTL-aligned GWAS results. Gene set enrichment analysis (GSEA) was then applied to identify BMD-associated gene sets with a predefined significance level of 0.05. Results We identified multiple gene sets associated with BMD in one or more regions, including relevant known biological gene sets such as the Reactome Circadian Clock (GSEA p-value = 1.0 × 10-4 for LS and 2.7 × 10-2 for femoral necks BMD in eQTLs-based GSEA) and insulin-like growth factor receptor binding (GSEA p-value = 5.0 × 10-4 for femoral necks and 2.6 × 10-2 for lumbar spines BMD in meQTLs-based GSEA). Conclusion Our results provided novel clues for subsequent functional analysis of bone metabolism, and illustrated the benefit of integrating eQTLs and meQTLs data into pathway association analysis for genetic studies of complex human diseases. Cite this article: W. Wang, S. Huang, W. Hou, Y. Liu, Q. Fan, A. He, Y. Wen, J. Hao, X. Guo, F. Zhang. Integrative analysis of GWAS, eQTLs and meQTLs data suggests that multiple gene sets are associated with bone mineral density. Bone Joint Res 2017;6:572–576.

Summary Objective To determine genome-wide DNA methylation profiles of knee cartilage from patients with Kashin-Beck disease (KBD) and osteoarthritis (OA). Method Knee cartilage was collected from 14 grade III KBD patients, 5 primary OA patients and 13 healthy subjects. The genome-wide methylation profiles of 5 KBD cartilage, 5 OA cartilage and 5 normal cartilage were determined by Illumina HumanMethylation450 array. Illumina Methylation Analyzer package was employed for identifying differentially methylated CpG sites. Functional annotation and enrichment analysis of differentially methylated genes (DMG) were conducted using GeneRIF database, Ingenuity Pathway Analysis (IPA) and The Database for Annotation, Visualization and Integrated Discovery (DAVID). Mass spectrometry (MS) and immunohistochemistry (IHC) were conducted to validate the functional relevance of identified KBD associated gene. Results We identified a total of 1212 differentially methylated CpG sites in KBD vs Normal, annotated to 264 hypermethylated and 368 hypomethylated genes. Comparing the DNA methylation profiles of KBD vs Normal and OA vs Normal detected overlap of 367 differentially methylated CpG sites (annotated to 182 genes) as well as 845 KBD-specific differentially methylated CpG sites (annotated to 471 unique genes). MS and IHC confirmed the hypermethylation status and decreased protein expression of HAPLN1 gene in KBD cartilage. Conclusion Our data implicate epigenetic dysregulation of a host of genes in KBD and OA. Furthermore, we observed common causal epigenetic changes shared by KBD and OA.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD(+) and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5′-dRP/AP lyase activity in PARP-1.

The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase beta (Pol beta). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE.Pol beta.DNA that formed specifically at BER intermediates containing a 5'-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol beta gap-filling and much stronger 5'-deoxyribose phosphate lyase activities than was observed with the Pol beta.DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol beta, although coordination also may be facilitated by APE.Pol beta.DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair.

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase β (Pol β) in complex with a 1-nt gapped DNA substrate containing a template N 2 -guanine adduct of the tumorigenic (−)-benzo[ c ]phenanthrene 4 R ,3 S -diol 2 S ,1 R -epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3′ to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol β insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol β also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol β-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.

This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl−1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.

H-FABP is regarded as a tissue-specific protein existing only in myocardial cells. It is released from the cardiac tissue and gets into the plasma when a heart attack occurs; the myocardial infarction is a good case in point. As a result, the detection of H-FABP will be an early and important biomarker for the disease concerned. The objective of the study is to prepare the recombinant H-FABP by aeukaryotic expression system, pichia , to produce the protein mimicking natural H-FABP, as an immunogen for the production of the specific antibody. A gene fragment encoding H-FABP was cloned in the expressing vector pPICZα, after sequencing. The recombinant plasmid was transformed into the competent cells of the X-33 strain by means of electroporation. The expression of the target peptide induced by methanol was screened by means of Western blotting, with the available MAb (Clone 6B6). Highly expressive engineer strains were obtained. The production of recombinant H-FABP under induction was about 0.7 g/L, with an M , of 14.5 kDa and recognized by a commercially available MAb (Clone 6B6). The recombinant vector was successfully constructed. Following this, H-FABP was expressed in X-33, and it would become the source of the preparation of specific antibodies, to develop diagnostic kits.

Mammalian DNA polymerase (pol) lambda is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol beta. Because pol beta provides two important enzymatic activities for base excision repair (BER), we examined whether pol lambda might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol beta and pol lambda. In combination with neutralizing antibodies against pol beta and pol lambda, our results show a BER deficiency in the pol lambda -/- cell extract compared with extract from isogenic wild-type cells. In addition, the pol lambda antibody strongly reduced in vitro BER in the pol beta -/- cell extract. These data indicate that pol lambda is able to contribute to BER in mouse fibroblast cell extract.

DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) are key players in pol beta-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol beta strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol beta DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5'-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol beta-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol beta substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol beta DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol beta. This study demonstrates that pol beta cooperates with FEN1 to remove DNA damage via a "Hit and Run" mechanism, involving alternating short gap production by FEN1 and gap filling by pol beta, rather than through coordinated formation and removal of a strand-displaced flap.

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase s (s-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3′- to 5′-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5′-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.

We have devised simplified protocols to purify large quantities of histidine-tagged (His-tagged) and untagged heterodimeric forms of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). Here, we report the optimization of overexpression and purification of heterodimeric RT expressed in Escherichia coli. The coding sequences of p66 and p51 subunits of RT were amplified using PCR from HXB2 HIV-1 and cloned into a bacterial expression system. The resulting expression plasmids for the RT subunits, pET-RT66 and pET-RT51, were under a strong T7/lac promoter that is induced by isopropyl-beta-d-thiogalactopyranoside. Purification of heterodimeric forms of RT was facilitated by high-level expression of these subunits that represented approximately 30-40% of total cell protein. For purification of the His-tagged heterodimeric RT, cell pellet from cells expressing the untagged p66 subunit was mixed in excess with a cell pellet expressing tagged p51. For untagged heterodimeric RT, the pellet from cells expressing p51 was mixed in excess with pellet expressing p66. Subunit dimerization occurred during cell lysis. During the subsequent chromatography steps, stable p66/p51 heterodimer was purified to homogeneity. The heterodimeric nature of the final preparations of RT was confirmed by analytical gel filtration, mass spectrometry, and denaturing gel electrophoresis. Further, the sensitivity of these enzyme preparations to AZTTP indicated that the histidine tag had no effect on nucleoside inhibitor binding, nucleotide binding or insertion, or DNA binding. The application of these expression/purification methodologies represents a useful method to purify large quantities of heterodimeric RT for structural investigations and provides an efficient protocol to produce subunit-specific amino acid alterations necessary for unambiguous structure/function investigations.

Human DNA polymerase iota (pol iota) is a member of the Y-family of low fidelity lesion bypass DNA polymerases. In addition to a probable role in DNA lesion bypass, this enzyme has recently been shown to be required for somatic hypermutation in human B-cells. We found earlier that human pol iota has deoxyribose phosphate (dRP) lyase activity and unusual specificity for activity during DNA synthesis, suggesting involvement in specialized forms of base excision repair (BER). Here, mapping of the domain structure of human pol iota by controlled proteolysis revealed that the enzyme has a 48-kDa NH2-terminal domain and a protease resistant 40-kDa "core domain" spanning residues Met79 to approximately Met445. A covalently cross-linked pol iota-DNA complex, representing a trapped intermediate in the dRP lyase reaction, was subjected to controlled proteolysis. Cross-linking was mapped to the 40-kDa core domain, indicating that the dRP lyase active site is in this region. To further evaluate the BER capacity of the enzyme, the dRP lyase and DNA polymerase activities were characterized on DNA substrates representing BER intermediates, and we found that pol iota was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase beta null cell extract.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.

The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N -methyl- N ′-nitro- N -nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.

This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mgl−1 BA and 1.0 mgl−1 NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mgl−1 NAA and 0.5 mgl−1 indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.

The objective of this study was to characterize the subgingival microbiota of African-American children with Localized Aggressive Periodontitis (LAP). Fifty-one children were included. Subgingival plaque samples were taken from diseased (DD) and healthy sites (DH) in LAP and from healthy sites in HS and HC and analyzed by 16S rRNA-based microarrays. Aggregatibacter actinomycetemcomitans (Aa) was the only species found to be both more prevalent (OR = 8.3, p = 0.0025) and abundant (p < 0.01) in DD. Filifactor alocis (Fa) was also found to be more prevalent in DD (OR 2.31, CI 1.06-5.01, p = 0.03). Most prevalent species in healthy sites were Selenomonas spp, Veillonella spp, Streptococcus spp, Bergeyella sp, and Kingella oralis. Overall, Streptococcus spp, Campylobacter gracilis, Capnocytophaga granulosa, Haemophilus parainfluenzae, and Lautropia mirabilis were most abundant in healthy children, while Aa, Fa, Tannerella sp, Solobacterium moorei, Parvimonas micra, and Capnocytophaga sp were most abundant in LAP. Based on a comprehensive analysis with 16S rRNA-based microarrays, Aa was strongly associated and site-specific in LAP. In contrast, other species were found to be associated with healthy sites and individuals (ClinicalTrials.gov number CT01330719). Abbreviations: healthy site in healthy sibling (HS); healthy site in healthy control child (HC).

Mouse cDNAs encoding ribosomal proteins (r-proteins), L12 and L18, were isolated and their sequences determined. The L12 cDNA was found to contain 639 bp, including a coding sequence of 498 nucleotides (nt), 5' (78 nt) and 3' (45 nt) untranslated regions (UTRs), and a poly(A) tail of 18 nt. The L18 cDNA was shown to consist of 648 bp, including a coding sequence of 567 nt, 5' (26 nt) and 3' (39 nt) UTRs, and a poly(A) tail of 16 nt. The nt sequences of the protein-coding region from the mouse L12 and L18 cDNAs were found to exhibit 96% and 92% identity, respectively, with those of the rat. With the use of mouse L12 and L18 cDNA probes, multiple (at least 10) copies of the L12 and L18 gene families were shown to be present in the mouse and rat genomes. However, there was no sequence heterogeneity detected among seven L18 cDNA clones, indicating that only one copy of the L18 gene-related sequences is functional, and the other copies are presumably nonfunctional pseudogenes. The complete amino acid (aa) sequences of the mouse r-proteins, L12 and L18, were deduced from the nt sequences of their cDNA clones. L12 has 165 aa and a Mr, of 17 790, while L18 has 188 aa and a Mr of 21 570. The aa sequences of the mouse r-proteins, L12 and L18, exhibit 98% and 94% identity, respectively, to those of rat.

5 pages; International audience; This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

Production of cellulose‐hydrogen from corn stalk based on acid‐enzyme two‐stage pretreatment by lesser panda manure was carried out in batch tests. The acid‐enzyme two‐stage pretreatment of corn stalk was found most effective, in which the yields of soluble saccharides (SS) were 470 mg/g‐TS. The maximum cumulative H2 yield (165.8 ml H2/g‐TS) and H2 production rate (12.8 ml H2/g‐TS h−1) were obtained at pH 5.5, 36 °C by treating a substrate of 15 g/L. The hydrogen content in biogas was 57.0% and there was no significant methane gas observed.

The oxidized DNA base 8-oxoguanine (8-oxoG) is implicated in neuronal CAG repeat expansion associated with Huntington disease, yet it is unclear how such a DNA base lesion and its repair might cause the expansion. Here, we discovered size-limited expansion of CAG repeats during repair of 8-oxoG in a wild-type mouse cell extract. This expansion was deficient in extracts from cells lacking pol beta and HMGB1. We demonstrate that expansion is mediated through pol beta multinucleotide gap-filling DNA synthesis during long-patch base excision repair. Unexpectedly, FEN1 promotes expansion by facilitating ligation of hairpins formed by strand slippage. This alternate role of FEN1 and the polymerase beta (pol beta) multinucleotide gap-filling synthesis is the result of uncoupling of the usual coordination between pol beta and FEN1. HMGB1 probably promotes expansion by stimulating APE1 and FEN1 in forming single strand breaks and ligatable nicks, respectively. This is the first report illustrating that disruption of pol beta and FEN1 coordination during long-patch BER results in CAG repeat expansion.

DNA polymerase theta (Pol theta) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol theta has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol theta has intrinsic 5'-deoxyribose phosphate (5'-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol theta is a approximately 300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol theta possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH(4) cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol theta. These results are consistent with a role of Pol theta in BER.

Loss of a tumor-suppressor gene function appears to play a critical role in the multistep process of neoplastic transformation of Syrian hamster embryo (SHE) cells in vitro. Clonal variants of two independent, preneoplastic cell lines have been isolated that have either retained (termed supB+) or lost (termed supB-) the ability to suppress the tumorigenicity of a highly malignant benzo[alpha]pyrene-transformed SHE cell line (BP6T) in cell hybrids. We have pursued several approaches in an attempt to identify genes that are responsible for tumor suppression in these cells. The only consistent differences detected in two-dimensional gel analyses of supB+ and supB- cellular proteins were decreases in the levels of two high molecular weight isoforms of tropomyosin in supB- cells. Differential screening of a supB+ cDNA library for genes that are preferentially expressed in supB+ cells yielded cDNA clones for four genes, i.e., collagen type II, collagen type IX, H19, and a previously unidentified gene (clone 5). Nuclear run-on assays suggested that higher transcription rates were responsible for the increased steady-state levels of some of these transcripts in supB+ cells. DNA sequence comparisons showed that two copies of a 9 bp element, previously identified in each of the mouse H19 enhancers, were also present in the 5' flanking region of the rat type II collagen gene. A transcription factor that controls expression of the collagen and H19 genes through binding to this conserved motif would be an attractive candidate for the supB+ gene or at least a mediator of the supB+ phenotype.

Summary Deoxyribose phosphate (dRP) removal by DNA polymerase β (Pol β) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol β null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1 −/− mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.

The human Amino Enhancer of Split (AES) gene encodes a protein of 197 amino acids exhibiting strong similarity to the N-terminal domain of Drosophila Enhancer of Split Groucho (ESG) protein. The nucleotide sequence of approximately 12 kb from the human AES gene was determined, and its protein-encoding sequence was shown to be interrupted by six introns. The human AES gene was further localized by fluorescence in situ hybridization to chromosome band 19p13.3 near the transcription factor 3 (TCF3) gene.

Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H2O2, but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLβ. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLβ, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLβ share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER. © 2006 Elsevier Inc. All rights reserved.

Vaccine-induced T cell responses to FIV were assessed by measuring FIV-specific cytokine and cytotoxic-effector molecule production. A total of 22 cats at 10-12 weeks of age received either dual-subtype FIV vaccine (n=12), uninfected cell lysate (n=5) consisting of cells used to produce vaccine viruses, or no immunization (n=5). Significant increases in mRNA and protein production of T-helper 1 (Th1) cytokines (IL-2, IFNgamma), mRNA production of a cytotoxic-effector molecule (perforin), and lymphoproliferation response were observed in peripheral blood mononuclear cells (PBMC) from dual-subtype FIV-vaccinated cats after in vitro stimulation with inactivated FIV. In contrast, no statistically significant increase in FIV-stimulated mRNA production of Th2 cytokines (IL-4, IL-6) or other cytotoxic-effector molecules (TNFalpha, FasL) was observed in the PBMC from dual-subtype vaccinated cats. Moreover, no FIV-specific increases in the IFNgamma, IL-2, and perforin mRNA productions and in the IFNgamma bioactivity and lymphoproliferation responses were observed in the PBMC from cell-immunized cats. These observations suggest that IFNgamma induction, lymphoproliferation, and significant portion of IL-2 and perforin productions in the PBMC from dual-subtype vaccinated cats are clearly specific for viral antigens. Overall, dual-subtype FIV vaccine elicited strong Th1 response (IFN(, IL-2), which may contribute to the vaccine protection by enhancing the perforin-mediated cytotoxic-cell activity against FIV.

The major product of oxidative base damage is 8-oxo-7,8-dihydro-2′-deoxyguanine (8odG). The coding potential of this lesion is modulated by its glycosidic torsion angle that controls whether its Watson-Crick or Hoogsteen edge is utilized for base pairing. The 2.0 Å structure of DNA polymerase (pol) β bound with 8odGTP opposite template adenine indicates that the modified nucleotide assumes the mutagenic syn-conformation and that the non-mutagenic anti-conformation would be incompatible with efficient DNA synthesis.

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.

Recent molecular cytogenetic studies have elucidated the origin and nature of extra structurally abnormal chromosomes (ESACs) or small supernumerary chromosomes, which are often associated with developmental delay and malformations. We studied the prevalence of inv dup(15) in a nationwide screening programme for mentally retarded children in Taiwan and tried to correlate the genotype and phenotype in those patients. Fluorescence in situ hybridization (FISH) analysis using D15Z, D15Z1, and the cosmids from the Prader-Willi/Angelman syndrome chromosome region (PW/ASCR) was performed on 54 patients (0.45%) with ESACs from 11893 probands within a 5-year period. Of them, inv dup(15) was confirmed in 25 children (46.3%) by FISH analysis. The PW/ASCR probes were used to clarify the size and DNA composition of the markers. Patients with inv dup(15) chromosomes, containing only the heterochromatin or little euchromatin of the proximal 15q (i.e., pter→q11:q11→pter) may have a rather mild or nearly normal phenotype (group 1). Only one patient had some features suggestive of Angelman syndrome, but was considered to be a result of deleted (15)(q12) in the chromosome 15 homologue. Additional copies within D15S11 through GABRB3 (15q11.2-13) resulted in an abnormal phenotype which involved mental and developmental delay but was different from the classical phenotype of PW/AS (groups 2, 3). Signs of autistic behavior did occur in each group. FISH combined with microsatellite analyses showed that the marker was often of maternal origin in de novo cases (n = 12, 86%), or inherited from the mother in only one familial case. Down-inv dup(15) was mentioned in two cases. Unusual features including diaphragmatic eventration, hyperlaxity of joints, arachnodactyly, brain atrophy, epilepsy (particularly infantile spasm), ataxia, genital abnormalities, and cleft lip/palate were noted in those patients. This observation expands the range of phenotypic expression associated with this relatively common ESAC. Conclusion Marked phenotypic diversities exist in children with inv dup(15), dependent upon the size or genetic composition of the markers, degree of mosaicism, parental origin and familial occurrence or not. Patients with a larger inv dup(15) marker chromosome including the PW/ASCR may have a higher risk of abnormalities, but not the typical Prade-Willi/Angelman syndrome phenotype.

We report a female baby with multiple congenital anomalies including left upper amelia, congenital short bowel with malrotation and pseudo-obstruction, dextrocardia with situs solitus, patent ductus arteriosus, and a tiny atrophic spleen. Chromosome study showed de novo 46,XX/46,XX,-4, + r(4)(p16-->q22.3)/47,XX,4, + r(4) (p16-->q22.3), + del(4)(pter-->q22.3:). The clinical findings in the patient were probably caused by the interaction of partial trisomy 4pter-->q22.3 or 4p16-->q22.3 and partial monosomy of 4q22.3-->4qter. This karyotype and phenotype have not previously been reported.

For many of the spinal cord-injured (SCI) men who are able to produce an ejaculate, infertility because of poor semen quality is a concern. Impaired spermatogenesis has been attributed as a possible reason for the poor semen quality. If so, events that occur during spermatogenesis may be used as a marker to evaluate the extent of spermatogenic alteration. During spermatogenesis, when the sperm nuclear condensation occurs, lysine-rich somatic histone are replaced by arginine-rich protamines in the DNA. Acidic aniline blue preferentially stains the immature sperm nucleus blue by binding to the lysine. Hence, each sperm can be individually evaluated for nuclear maturity. To test this concept, the nuclear maturity of sperm from 12 SCI men obtained by vibratory stimulation was compared with sperm samples obtained by self-masturbation from 104 non-SCI men. Sperm smears stained with acidic aniline blue were evaluated for nuclear maturity. The percent of unstained spermatozoa for non-SCI men (mean +/- SEM; 83.4 +/- 1.1%) was not statistically different from that of the SCI men (79.7 +/- 4.8%). However, the sperm motility (70.5 +/- 1.2%) and the percentage of normal sperm morphology (50.8 +/- 0.7%) of non-SCI men were significantly (P.01) different from those of the SCI men (36.5 +/- 6.8% and 44.0 +/- 2.4%). It seems that the poor semen quality observed in SCI men is probably not caused by inadequate nuclear maturity of the spermatozoa.

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

Mouse and human cDNA encoding AES (amino-terminal enhancer of split) and ESG (enhancer of split groucho) proteins with strong similarity to Drosophila enhancer of split groucho protein were isolated and sequenced. Mouse AES-1 and AES-2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins were found to exhibit approximately 50% identity to the amino-terminal region of Drosophila groucho, mouse ESG and human transducin-like enhancer of split (TLE) proteins. Mouse AES transcripts of 1.5 kb and 1.2 kb were abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 kb and 1.4 kb were predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3 kb and 4.0 kb were found only in testis, while human TLE 1 transcripts of 4.5 kb was more abundant in muscle and placenta compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosomes 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines.

Mammalian base excision repair (BER) is mediated through at least two subpathways designated 'single-nucleotide' (SN) and 'long-patch' (LP) BER (2-nucleotides long/more repair patch). Two forms of DNA substrate are generally used for in vitro BER assays: oligonucleotide- and plasmid-based. For plasmid-based BER assays, the availability of large quantities of substrate DNA with a specific lesion remains the limiting factor. Using sequence-specific endonucleases that cleave only one strand of DNA on a double-stranded DNA substrate, we prepared large quantities of plasmid DNA with a specific lesion. We compared the kinetic features of BER using plasmid and oligonucleotide substrates containing the same lesion and strategic restriction sites around the lesion. The K(m) for plasmid DNA substrate was slightly higher than that for the oligonucleotide substrate, while the V(max) of BER product formation for the plasmid and oligonucleotide substrates was similar. The catalytic efficiency of BER with the oligonucleotide substrate was slightly higher than that with the plasmid substrate. We conclude that there were no significant differences in the catalytic efficiency of in vitro BER measured with plasmid and oligonucleotide substrates. Analysis of the ratio of SN BER to LP BER was addressed using cellular extracts and a novel plasmid substrate.

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.

The cloned cDNA encoding for mouse sperm-specific lactate dehydrogenase C (LDH-C) was inserted immediately downstream to the MMTV 5′ LTR promoter, and it was shown to synthesize mouse LDH-C polypeptide in Chinese-hamster ovary cells. The mouse LDH-C subunit and the endogenous Chinese-hamster LDH-A subunit formed in vivo a heterotetrameric LDH-A3C1 isoenzyme, and this novel isoenzyme exhibited enzymic activity utilizing lactate as substrate.

The promoter region of mouse lactate dehydrogenase-A gene was fused with the chloramphenicol acetyltransferase gene of Escherichia coli , and the expression of this fusion gene was induced by cyclic AMP in Chinese hamster ovary wild-type cells, but not in mutants affecting the regulatory or catalytic subunit of cAMP-dependent protein kinase.

The cloned cDNA encoding for human lactate dehydrogenase-A was inserted immediately downstream to the SV40 early promoter, and it was shown to synthesize the human LDH-A polypeptide in Chinese hamster ovary cells. The human LDH-A subunit and the endogenous Chinese hamster LDH-a subunit formed in vivo a heterotetrameric LDH-a3A1 functional isoenzyme, indicating the conserved tertiary structure of both LDH-A subunits.