Decadal warming causes a consistent and persistent shift from heterotrophic to autotrophic respiration in contrasting permafrost ecosystems

Soil carbon in permafrost ecosystems has the potential to become a major positive feedback to climate change if permafrost thaw increases heterotrophic decomposition. However, warming can also stimulate autotrophic production leading to increased ecosystem carbon storage—a negative climate change feedback. Few studies partitioning ecosystem respiration examine decadal warming effects or compare responses among ecosystems. Here, we first examined how 11 years of warming during different seasons affected autotrophic and heterotrophic respiration in a bryophyte‐dominated peatland in Abisko, Sweden. We used natural abundance radiocarbon to partition ecosystem respiration into autotrophic respiration, associated with production, and heterotrophic decomposition. Summertime warming decreased the age of carbon respired by the ecosystem due to increased proportional contributions from autotrophic and young soil respiration and decreased proportional contributions from old soil. Summertime warming's large effect was due to not only warmer air temperatures during the growing season, but also to warmer deep soils year‐round. Second, we compared ecosystem respiration responses between two contrasting ecosystems, the Abisko peatland and a tussock‐dominated tundra in Healy, Alaska. Each ecosystem had two different timescales of warming (<5 years and over a decade). Despite the Abisko peatland having greater ecosystem respiration and larger contributions from heterotrophic respiration than the Healy tundra, both systems responded consistently to short‐ and long‐term warming with increased respiration, increased autotrophic contributions to ecosystem respiration, and increased ratios of autotrophic to heterotrophic respiration. We did not detect an increase in old soil carbon losses with warming at either site. If increased autotrophic respiration is balanced by increased primary production, as is the case in the Healy tundra, warming will not cause these ecosystems to become growing season carbon sources. Warming instead causes a persistent shift from heterotrophic to more autotrophic control of the growing season carbon cycle in these carbon‐rich permafrost ecosystems.     

Drainage enhances modern soil carbon contribution but reduces old soil carbon contribution to ecosystem respiration in tundra ecosystems

Warming temperatures are likely to accelerate permafrost thaw in the Arctic, potentially leading to the release of old carbon previously stored in deep frozen soil layers. Deeper thaw depths in combination with geomorphological changes due to the loss of ice structures in permafrost, may modify soil water distribution, creating wetter or drier soil conditions. Previous studies revealed higher ecosystem respiration rates under drier conditions, and this study investigated the cause of the increased ecosystem respiration rates using radiocarbon signatures of respired CO₂ from two drying manipulation experiments: one in moist and the other in wet tundra. We demonstrate that higher contributions of CO₂ from shallow soil layers (0–15 cm; modern soil carbon) drive the increased ecosystem respiration rates, while contributions from deeper soil (below 15 cm from surface and down to the permafrost table; old soil carbon) decreased. These changes can be attributed to more aerobic conditions in shallow soil layers, but also the soil temperature increases in shallow layers but decreases in deep layers, due to the altered thermal properties of organic soils. Decreased abundance of aerenchymatous plant species following drainage in wet tundra reduced old carbon release but increased aboveground plant biomass elevated contributions of autotrophic respiration to ecosystem respiration. The results of this study suggest that drier soils following drainage may accelerate decomposition of modern soil carbon in shallow layers but slow down decomposition of old soil carbon in deep layers, which may offset some of the old soil carbon loss from thawing permafrost.     

Carbon (δ13C) and nitrogen (δ15N) stable isotope composition in plant and soil in Southern Patagonia's native forests

Stable isotope natural abundance measurements integrate across several biogeochemical processes in ecosystem N and C dynamics. Here, we report trends in natural isotope abundance (δ¹³C and δ¹⁵N in plant and soil) along a climosequence of 33 Nothofagus forest stands located within Patagonia, Southern Argentina. We measured 28 different abiotic variables (both climatic variables and soil properties) to characterize environmental conditions at each of the 33 sites. Foliar δ¹³C values ranged from ?35.4‰ to ?27.7‰, and correlated positively with foliar δ¹⁵N values, ranging from ?3.7‰ to 5.2‰. Soil δ¹³C and δ¹⁵N values reflected the isotopic trends of the foliar tissues and ranged from ?29.8‰ to ?25.3‰, and ?4.8‰ to 6.4‰, respectively, with no significant differences between Nothofagus species (Nothofagus pumilio, Nothofagus antarctica, Nothofagus betuloides). Principal component analysis and multiple regressions suggested that mainly water availability variables (mean annual precipitation), but not soil properties, explained between 42% and 79% of the variations in foliar and soil δ¹³C and δ¹⁵N natural abundance, which declined with increased moisture supply. We conclude that a decline in water use efficiency at wetter sites promotes both the depletion of heavy C and N isotopes in soil and plant biomass. Soil δ¹³C values were higher than those of the plant tissues and this difference increased as annual precipitation increased. No such differences were apparent when δ¹⁵N values in soil and plant were compared, which indicates that climatic differences contributed more to the overall C balance than to the overall N balance in these forest ecosystems.     

Partitioning respiration of C3-C4 mixed communities using the natural abundance 13C approach--testing assumptions in a controlled environment

Contributions of C3 and C4 plants to respiration of C3-C4 ecosystems can be estimated on the basis of their contrasting 13C discrimination. But accurate partitioning requires accurate measurements of the isotope signature of whole system respiratory CO2 (deltaR), and of its members (delta3 and delta4). Unfortunately, experimental determination of representative delta3 and delta4 values is virtually impossible in nature, generating a need for proxies (surrogates) of delta3 and delta4 values (e.g., the delta of leaf biomass). However, recent evidence indicates that there may be systematic differences among the delta of respiratory and biomass components. Thus, partitioning may be biased depending on the proxy. We tested a wide range of biomass- and respiration-based delta proxies for the partitioning of respiration of mixed Lolium perenne (C3) - Paspalum dilatatum (C4) stands growing at two temperatures inside large 13CO2/ 12CO2 gas exchange chambers. Proxy-based partitioning was compared with results of reference methods, including (i) the delta of whole plant respiratory CO2 (delta3 and delta4) or (ii) respiration rate of intact C3 and C4 plants. Results of the reference methods agreed near perfectly. Conversely, some proxies yielded erroneous partitioning results. Partitioning based on either the delta of shoot or root respiratory CO2 produced the worst bias, because shoot respiratory CO2 was enriched in 13C by several per thousand and root respiratory CO2 was depleted by several per thousand relative to whole plant respiratory CO2. Use of whole plant or whole shoot biomass delta gave satisfactory partitioning results under the constant conditions of the experiments, but their use in natural settings is cautioned if environmental conditions are variable and the time scales of respiration partitioning differ strongly from the residence time of C in biomass. Other biomass-based proxies with faster turnover (e.g., leaf growth zones) may be more useful in changing conditions.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Annual variability in dentin δ15N and δ13C reveal sex differences in weaning age and feeding habits in Risso's dolphins (Grampus griseus)

Teeth of odontocetes accumulate annual dentinal growth layer groups (GLGs) that record isotope ratios, which reflect the time of their synthesis. Collectively, they provide lifetime records of individual feeding patterns from which life history traits can be inferred. We subsampled the prenatal dentin and postnatal GLGs in Risso's dolphins (Grampus griseus) (n = 65) that stranded or were collected as bycatch in Taiwan (1994–2014) and analyzed them for δ¹⁵N and δ¹³C. Age‐specific δ¹⁵N and δ¹³C values were corrected for effects of calendar year, stranding site, C/N, and sex. δ¹⁵N values were higher in prenatal layers (14.94‰ ± 0.74‰) than in adult female GLGs (12.58‰ ± 0.20‰), suggesting fetal enrichment during gestation. Decreasing δ¹⁵N values in early GLGs suggested changes in dietary protein sources during transition to complete weaning. Weaning age was earlier in males (1.09 yr) than in females (1.81 yr). Significant differences in δ¹⁵N values between weaned males and females suggest potential sexual segregation in feeding habits. δ¹³C values increased from the prenatal to the 4th GLG by ~1.0‰, indicative of a diet shift from ¹³C‐depleted milk to prey items. Our results provide novel insights into the sex‐specific ontogenetic changes in feeding patterns and some life history traits of Risso's dolphins.     

Moss δ13C: an accurate proxy for past water environments in polar regions

Increased aridity is of global concern. Polar regions provide an opportunity to monitor changes in bioavailable water free of local anthropogenic influences. However, sophisticated proxy measures are needed. We explored the possibility of using stable carbon isotopes in segments of moss as a fine‐scale proxy for past bioavailable water. Variation in δ¹³C with water availability was measured in three species across three peninsulas in the Windmill Islands, East Antarctica and verified using controlled chamber experiments. The δ¹³C from Antarctic mosses accurately recorded long‐term variations in water availability in the field, regardless of location, but significant disparities in δ¹³C between species indicated some make more sensitive proxies. δ¹³C_SUGAR derived from living tissues can change significantly within the span of an Antarctic season (5 weeks) in chambers, but under field conditions, slow growth means that this technique likely represents multiple seasons. δ¹³C_CELLULOSE provides a precise and direct proxy for bioavailable water, allowing reconstructions for coastal Antarctica and potentially other cold regions over past centuries.     

Extreme rainfall and snowfall alter responses of soil respiration to nitrogen fertilization: a 3‐year field experiment

Extreme precipitation is predicted to be more frequent and intense accompanying global warming and may have profound impacts on soil respiration (Rs) and its components, that is, autotrophic (Ra) and heterotrophic (Rh) respiration. However, how natural extreme rainfall or snowfall events affect these fluxes are still lacking, especially under nitrogen (N) fertilization. In this study, extreme rainfall and snowfall events occurred during a 3‐year field experiment, allowing us to examine their effects on the response of Rs, Rh, and Ra to N supply. In normal rainfall years of 2011/2012 and 2012/2013, N fertilization significantly stimulated Rs by 23.9% and 10.9%, respectively. This stimulation was mainly due to the increase of Ra because of N‐induced increase in plant biomass. In the record wet year of 2013/2014, however, Rs was independent on N supply because of the inhibition effect of the extreme rainfall event. Compared with those in other years, Rh and Ra were reduced by 36.8% and 59.1%, respectively, which were likely related to the anoxic stress on soil microbes and decreased photosynthates supply. Although N supply did not affect annual Rh, the response ratio (RR) of Rh flux to N fertilization decreased firstly during growing season, increased in nongrowing season and peaked during spring thaw in each year. Nongrowing season Rs and Rh contributed 5.5–16.4% to their annual fluxes and were higher in 2012/2013 than other years due to the extreme snowfall inducing higher soil moisture during spring thaw. The RR of nongrowing season Rs and Rh decreased in years with extreme snowfall or rainfall compared to those in normal years. Overall, our results highlight the significant effects of extreme precipitation on responses of Rs and its components to N fertilization, which should be incorporated into models to improve the prediction of carbon‐climate feedbacks.     

VARIATION IN INTERNAL δ15N AND δ13C DISTRIBUTIONS AND THEIR BULK VALUES IN THE BROWN MACROALGA PADINA AUSTRALIS GROWING IN SUBTROPICAL OLIGOTROPHIC WATERS1

The utility of δ¹⁵N measurements in Padina australis Hauck as a probe for its external nitrogen (N) sources was tested by monitoring the bulk values of chemical components [δ¹⁵N, δ¹³C, and N and carbon (C) contents] and their internal distributions during a 12 d incubation in a controlled environment. Under the saturated conditions of isotopically heavier nitrate than that of original algal tissue, the bulk δ¹⁵N in P. australis was enriched, but less than what was predicted from a simple mixing model, signaling possible isotopic discrimination during N assimilation and subsequent N efflux from the cells. The enhanced N content (%), which occurred simultaneously with this δ¹⁵N shift, was a useful signal indicating this phenomenon. Bulk δ¹⁵N was enriched, especially around the meristem, in tissues growing under conditions of higher irradiance and temperature, probably due in part to dissolved organic nitrogen (DON) excretion. The δ¹³C enhancement in bulk algal tissues, also associated with high photosynthetic activity, may be an additional signal indicating this unbalanced internal δ¹⁵N distribution. However, in summer and winter environmental conditions with periodic nitrate supplies simulating typical fringing reef waters, the difference in measured algal bulk δ¹⁵N from theoretical predictions was within ±1.0‰. This difference is very small compared with the variation in δ¹⁵N in possible N sources in coastal areas. In the field, therefore, δ¹⁵N in Padina can be used effectively to trace N sources in both space and time after determining algal N content and δ¹³C to determine whether large alterations occur in algal δ¹⁵N.     

Losses of soil carbon by converting tropical forest to plantations: erosion and decomposition estimated by δ13C

Indonesia lost more tropical forest than all of Brazil in 2012, mainly driven by the rubber, oil palm, and timber industries. Nonetheless, the effects of converting forest to oil palm and rubber plantations on soil organic carbon (SOC) stocks remain unclear. We analyzed SOC losses after lowland rainforest conversion to oil palm, intensive rubber, and extensive rubber plantations in Jambi Province on Sumatra Island. The focus was on two processes: (1) erosion and (2) decomposition of soil organic matter. Carbon contents in the Ah horizon under oil palm and rubber plantations were strongly reduced up to 70% and 62%, respectively. The decrease was lower under extensive rubber plantations (41%). On average, converting forest to plantations led to a loss of 10 Mg C ha^?1 after about 15 years of conversion. The C content in the subsoil was similar under the forest and the plantations. We therefore assumed that a shift to higher δ¹³C values in plantation subsoil corresponds to the losses from the upper soil layer by erosion. Erosion was estimated by comparing the δ¹³C profiles in the soils under forest and under plantations. The estimated erosion was the strongest in oil palm (35 ± 8 cm) and rubber (33 ± 10 cm) plantations. The ¹³C enrichment of SOC used as a proxy of its turnover indicates a decrease of SOC decomposition rate in the Ah horizon under oil palm plantations after forest conversion. Nonetheless, based on the lack of C input from litter, we expect further losses of SOC in oil palm plantations, which are a less sustainable land use compared to rubber plantations. We conclude that δ¹³C depth profiles may be a powerful tool to disentangle soil erosion and SOC mineralization after the conversion of natural ecosystems conversion to intensive plantations when soils show gradual increase of δ¹³C values with depth.     

Stable forest–savanna mosaic in north‐western Tanzania: local‐scale evidence from δ13C signatures and 14C ages of soil fractions

Aim The spatio‐temporal dynamics of dry evergreen forest patches in the savanna biome of the Kagera region (north‐western Tanzania) are largely unknown owing to a lack of pollen and macrofossil evidence. Our aims were to reconstruct local‐scale shifts of the forest–savanna boundary in order to determine whether the forests have been expanding or retreating on a centennial and millennial time‐scale. Location The Kagera region of north‐western Tanzania, East Africa. Methods The vegetation reconstruction was based on analysing δ¹³C signatures in soils along a transect spanning both C₄ open savanna and C₃ forest vegetation. Furthermore, we fractionated soil organic matter (SOM) according to density and chemical stability to analyse δ¹³C values of soil fractions with distinct radiocarbon ages. Results We found sharp changes in δ¹³C signatures in bulk SOM from the forest to the savanna, within a few metres along the transect. The forest soil profiles carried a persistent C₃‐dominated signature. Radiocarbon dating of the oldest, most recalcitrant forest soil fraction yielded a mean age of 5500 cal. yr bp , demonstrating that the forest has existed since at least the mid‐Holocene. The savanna sites showed a typical C₄ isotopic signature in SOM of topsoils, but subsoils and more recalcitrant SOM fractions also contained signals of C₃ plants. The dense soil fraction (ρ > 1.6 g cm^?3) carrying a pure C₄ label had a mean age of c. 1200 cal. yr bp , indicating the minimum duration of the dominance of grass vegetation on the savanna site. At the forest edge, the older C₄ grass signature of SOM has steadily been replaced by the more negative δ¹³C fingerprint of the forest trees. As this replacement has occurred mainly in the 10‐m‐wide forest–savanna ecotone over the last c. 1200 years, the forest expansion must be very slow and is very likely less than 15 m century^?1. Main conclusions Our results suggest that forest patches in the Kagera savanna landscape are very stable vegetation formations which have persisted for millennia. During the last millennium, they have been expanding very slowly into the surrounding savanna at a rate of less than 15 m century^?1.     

β-Agonists as Antiobesity,Antidiabetic and Nutrient Partitioning Agents

In the past decade, the antiobesity, antidiabetic and nutrient partitioning activities of β-agonists have been extensively studied. The data generated from these compounds in experimental and farm animals have convincingly proved that body fat content and body weight can be modified to some degree by a metabolic agent without decreasing food consumption. Marginal antiobesity and antidiabetic activities in humans have been demonstrated with a few mixed β-agonists under certain conditions, but their utility is limited by side effects. The concept of a β₃-receptor rose from the study of these compounds and has been verified by the cloning and expression of this receptor from several species. Rat β₃-selective agonists have so far shown no antiobesity efficacy in humans. The resolution of several issues is critical for the discovery and development of efficacious antiobesity and antidiabetic agents with minimum side effects. Ultimately, the further investigation of these β-agonists and β-receptors should lead to a better understanding of the relationship between energy metabolism and feeding behavior.     

Permafrost collapse alters soil carbon stocks,respiration, CH4, and N2O in upland tundra

Release of greenhouse gases from thawing permafrost is potentially the largest terrestrial feedback to climate change and one of the most likely to occur; however, estimates of its strength vary by a factor of thirty. Some of this uncertainty stems from abrupt thaw processes known as thermokarst (permafrost collapse due to ground ice melt), which alter controls on carbon and nitrogen cycling and expose organic matter from meters below the surface. Thermokarst may affect 20–50% of tundra uplands by the end of the century; however, little is known about the effect of different thermokarst morphologies on carbon and nitrogen release. We measured soil organic matter displacement, ecosystem respiration, and soil gas concentrations at 26 upland thermokarst features on the North Slope of Alaska. Features included the three most common upland thermokarst morphologies: active‐layer detachment slides, thermo‐erosion gullies, and retrogressive thaw slumps. We found that thermokarst morphology interacted with landscape parameters to determine both the initial displacement of organic matter and subsequent carbon and nitrogen cycling. The large proportion of ecosystem carbon exported off‐site by slumps and slides resulted in decreased ecosystem respiration postfailure, while gullies removed a smaller portion of ecosystem carbon but strongly increased respiration and N₂O concentration. Elevated N₂O in gully soils persisted through most of the growing season, indicating sustained nitrification and denitrification in disturbed soils, representing a potential noncarbon permafrost climate feedback. While upland thermokarst formation did not substantially alter redox conditions within features, it redistributed organic matter into both oxic and anoxic environments. Across morphologies, residual organic matter cover, and predisturbance respiration explained 83% of the variation in respiration response. Consistent differences between upland thermokarst types may contribute to the incorporation of this nonlinear process into projections of carbon and nitrogen release from degrading permafrost.     

Fast response of Scots pine to improved water availability reflected in tree‐ring width and δ13C

Drought‐induced forest decline, like the Scots pine mortality in inner‐Alpine valleys, will gain in importance as the frequency and severity of drought events are expected to increase. To understand how chronic drought affects tree growth and tree‐ring δ¹³C values, we studied mature Scots pine in an irrigation experiment in an inner‐Alpine valley. Tree growth and isotope analyses were carried out at the annual and seasonal scale. At the seasonal scale, maximum δ¹³C values were measured after the hottest and driest period of the year, and were associated with decreasing growth rates. Inter‐annual δ¹³C values in early‐ and latewood showed a strong correlation with annual climatic conditions and an immediate decrease as a response to irrigation. This indicates a tight coupling between wood formation and the freshly produced assimilates for trees exposed to chronic drought. This rapid appearance of the isotopic signal is a strong indication for an immediate and direct transfer of newly synthesized assimilates for biomass production. The fast appearance and the distinct isotopic signal suggest a low availability of old stored carbohydrates. If this was a sign for C‐storage depletion, an increasing mortality could be expected when stressors increase the need for carbohydrate for defence, repair or regeneration.     

Interpretation of soil δ13C as an indicator of vegetation change in African savannas

Question: The relationship between carbon‐13 in soil organic matter and C₃ and C₄ plant abundance is complicated because of differential productivity, litter fall and decomposition. As a result, applying a mass balance equation to δ¹³C data from soils cannot be used to infer past C₃ and C₄ plant abundance; only the proportion of carbon derived from C₃ and C₄ plants can be estimated. In this paper, we compare δ¹³C of surface soil samples with vegetation data, in order to establish whether the ratio of C₃:C₄ plants (rather than the proportion of carbon from C₃ and C₄ plants) can be inferred from soil δ¹³C. Location: The Tsavo National Park, in southeastern Kenya. Methods: We compare vegetation data with δ¹³C of organic matter in surface soil samples and derive regression equations relating the δ¹³C of soil organic matter to C₃:C₄ plant abundance. We use these equations to interpret δ¹³C data from soil profiles in terms of changes in inferred C₃:C₄ plant ratio. We compare our method of interpretation with that derived from a mass balance approach. Results: There was a statistically significant, linear relationship between the δ¹³C of organic matter in surface soil samples and the natural logarithm of the ratio of C₃:C₄ plants in the 100m² surrounding the soil sample. Conclusions: We suggest that interpretation of δ¹³C data from organic matter in soil profiles can be improved by comparing vegetation surveys with δ¹³C of organic matter in surface soil samples. Our results suggest that past C₃ plant abundance might be under‐estimated if a mass balance approach is used.     

Modeling of α(1–4) chain arrangements in α(1–4)(1–6) glucans: The action and outcome of β-amylase and Pseudomonas stutzeri amylase on an α(1–4)(1–6) glucan model

Simulated enzymic debranching of a β-limit dextrin model, prepared from a computed construct made by random extension and branching, and given the CCL value of w-maize amylopectin (and equal amounts of external chains with ECL values of 2 and 3) has been related to experimental chromatograms of the debranched β-limit dextrin of the amylopectin. The profile was similar to those from gel chromatograms and IEC-PAD chromatography.The equivalent lengths in glucosyl units of grid-links (g-links) of internal and external chains in constructs were calculated from the ICL and ECL values of amylopectin and models produced from the constructs with the appropriate lengths for internal and external chains. These derived models were subjected to simulated hydrolysis by Pseudomonas stutzeri amylase and the products compared with those of the experimental distribution from w-maize amylopectin. With the model the amounts of maltotetraose and maltodextrins released were similar to the experimental values but the distribution of branched maltodextrins was quite different. Unlike w-maize amylopectin – a polymer with the cluster structure – which has given a profile of molecular sizes of maltodextrins with low amounts of single and small numbers of internal chains and with a peak at a M_W of about 14,000 (13 chains), in the model the proportion of maltodextrin with one internal chain was high and as d.p. increased the amounts decreased exponentially. This would be expected if the distribution of internal chains in the core was random. It is suggested that in the core of a model prepared from a construct made with alternating probabilities of extension – one in which this probability is high relative to branching, and a second in which it is low – may give clusters of branched maltodextrins with short internal chains which are joined by longer chains; more closely approximating the distribution of internal chains of different lengths in amylopectin.An arrangement for amylopectin molecules in the starch granule has been proposed. In this, they have a wafer-like, discoidal shape, composed of the amorphous zone overlain with the double helical, crystalline region. The flat macromolecules are concentrically layered with the former on the inside and the latter oriented to the outside of the granule.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.