The 2B subdomain of Rep helicase links translocation along DNA with protein displacement

Helicases catalyse DNA and RNA strand separation. Proteins bound to the nucleic acid must also be displaced in order to unwind DNA. This is exemplified by accessory helicases that clear protein barriers from DNA ahead of advancing replication forks. How helicases catalyse DNA unwinding is increasingly well understood but how protein displacement is achieved is unclear. Escherichia coli Rep accessory replicative helicase lacking one of its four subdomains, 2B, has been shown to be hyperactivated for DNA unwinding in vitro but we show here that RepΔ2B is, in contrast, deficient in displacing proteins from DNA. This defect correlates with an inability to promote replication of protein-bound DNA in vitro and lack of accessory helicase function in vivo. Defective protein displacement is manifested on double-stranded and single-stranded DNA. Thus binding and distortion of duplex DNA by the 2B subdomain ahead of the helicase is not the missing function responsible for this deficiency. These data demonstrate that protein displacement from DNA is not simply achieved by helicase translocation alone. They also imply that helicases may have evolved different specific features to optimise DNA unwinding and protein displacement, both of which are now recognised as key functions in all aspects of nucleic acid metabolism.

SP526TREATMENT WITH PCSK9 INHIBITORS IN PERITONEAL DIALYSIS

Phylogeny, Distribution, and Pathogenicity of Lasiodiplodia Species Associated With Cankers and Dieback Symptoms of Persian Lime in Mexico

Persian lime ( Citrus latifolia Tan.) is an important and widely cultivated fruit crop in several regions of Mexico. In recent years, severe symptoms of gummosis, stem cankers, and dieback were detected in the Persian lime-producing region in the states of Veracruz and Puebla, Mexico. The aims of this study were to identify the species of Lasiodiplodia associated with these symptoms, determine the distribution of these species, and test their pathogenicity and virulence on Persian lime plants. In 2015, symptomatic samples were collected from 12 commercial Persian lime orchards, and 60 Lasiodiplodia isolates were obtained. Fungal identification of 32 representative isolates was performed using a phylogenetic analysis based on DNA sequence data of the internal transcribed spacer region and part of the translation elongation factor 1-α and β-tubulin genes. Sequence analyses were carried out using the Maximum Likelihood and Bayesian Inference methods. Six Lasiodiplodia species were identified as Lasiodiplodia pseudotheobromae , Lasiodiplodia theobromae , Lasiodiplodia brasiliense , Lasiodiplodia subglobosa , Lasiodiplodia citricola , and Lasiodiplodia iraniensis . All Lasiodiplodia species of this study are reported for the first time in association with Persian lime in Mexico and worldwide. L. pseudotheobromae (46.9% of isolates) was the most frequently isolated species followed by L. theobromae (28.1%) and L. brasiliense (12.5%). Pathogenicity on Persian lime young plants using a mycelial plug inoculation method showed that all identified Lasiodiplodia species were able to cause necrotic lesions and gummosis, bu L. subglobosa , L. iraniensis , and L. pseudotheobromae were the most virulent.

First Report of Powdery Mildew of European Plum Caused by Podosphaera tridactyla in Mexico

Effect of Dietary Sodium Intake on Blood Pressure Variability

Purpose Studies in rodents have demonstrated that increased dietary sodium intake elevates blood pressure variability (BPV), independent of changes in resting blood pressure. Clinical studies have shown that increased BPV independently contributes to target organ damage and cardiovascular events. However, the effects of high dietary sodium intake on BPV in young, normotensive adults remains unknown. Therefore, the purpose of this study was to test the hypothesis that increased dietary sodium intake alters BPV in young, healthy adults. Methods Fourteen healthy, normotensive adults (7M/7W; age: 25±4 yrs; BMI: 24.7±0.9 kg/m 2 , Mean‐±SEM) participated in a controlled feeding study that consisted of 10 days of either low‐(LS:1 g sodium/day), medium‐ (MS: 2.3 g sodium/day), or high‐sodium (HS: 7 g sodium/day) diets, in randomized order. The diets were separated by at least a month. Urinary sodium excretion was determined to ensure compliance on diets. Resting beat‐to‐beat BPV was assessed in the laboratory in the supine position for ten minutes. BPV was also derived from 24‐hour ambulatory blood pressure monitoring (ABPM) to assess BPV under free‐living conditions. BPV was calculated as the average real variability index (ARV; the average of the absolute differences between consecutive blood pressure measurements) and as standard deviation (SD). Results Twenty‐four‐hour urinary sodium excretion increased in a step‐wise manner from low, to the medium, to the high sodium diets (LS: 34±6 mmol/24 hours, MS: 93±9 mmol/24, HS:275±29 mmol/24 hours, p0.05) or 24h ABPM systolic ARV (LS=9.4±0.5 mmHg, MS= 9.9±0.4 mmHg, HS=10.3±0.7 mmHg, p>0.05). Dietary sodium also did not alter laboratory beat‐to‐beat systolic SD (LS= 5.4±0.5 mmHg, MS=5.1±0.3 mmHg, HS=5.8±0.6 mmHg, p>0.05) or 24h ABPM systolic SD (LS= 12.1±0.9 mmHg, MS= 13.9±0.7 mmHg, HS= 13.1±0.9 mmHg, p>0.05). Conclusion These preliminary data suggest that 10 days of high dietary sodium intake does not alter systolic BPV in young, healthy adults. Support or Funding Information NIH Grant 1RO1HL128388 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Effect of Short‐term Water Restriction on Blood Pressure Variability in Young Adults

Water restriction causes an acute decline in plasma volume and subsequently elevates serum sodium and plasma osmolality. Rodent models demonstrate that elevated serum sodium and plasma osmolality increases blood pressure (BP) variability. This is important because elevated BP variability is an independent risk factor for adverse cardiovascular events. However, the effects of water restriction on BP variability in humans remains to be determined. PURPOSE To test the hypothesis that short‐term water restriction increases serum sodium and plasma osmolality and leads to greater BP variability in humans. METHODS Young healthy adults (13M/11F, age: 25±1 yrs; BMI: 24±1 kg/m 2 ; BP 107±2/60±1 mmHg) completed two three‐day‐long hydration protocols in randomized order, separated by at least one week. Women were tested in the early follicular phase of their menstrual cycle. Daily water intake for euhydration (EU) was 23 mL H 2 O/kg/day. Water intake for hypohydration (HYPO) consisted of a stepwise daily reduction in water intake, followed by a 16‐hour water abstention period prior to testing. Laboratory testing was conducted on day four of the protocols. Participants wore an ambulatory BP monitor and collected urine for urinalysis for 24 hours preceding each study visit. The ambulatory BP monitoring provides BP variability under free‐living conditions. Upon arrival to the laboratory, participants rested quietly in a dimly lit room in the supine position for ten minutes for the assessment of beat‐to‐beat BP variability via finger photoplethysmography under controlled conditions. The average real variability (ARV) index, the average of the absolute differences between all consecutive BP measurements, was determined. Standard deviation (SD) of BP was also determined. RESULTS HYPO led to higher plasma osmolality (290.6±1.0 vs. 287.7±1.0 mOsm/kg H 2 O, p0.05 for both). Ambulatory mean and diastolic BP variability were not different between conditions (p>0.05). Beat‐to‐beat systolic BP variability was not different between HYPO and EU (ARV BP 2.0±0.1 vs. 1.9±0.1; SD BP 5.4±0.4 vs. 5.0±0.3, p>0.05 for both). Beat‐to‐beat mean and diastolic BP variability were also not different between conditions (p>0.05). CONCLUSION These preliminary data suggest that short‐term water restriction in young healthy adults does not increase ambulatory or beat‐to‐beat BP variability. Support or Funding Information Supported by NIH Grant 1R01HL128388 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Differential Sympathetic and RAAS Responses to a Low Sodium Diet

A common sodium recommendation intake target is 2300 mg/d; however the AHA has stated that no more than 1500 mg per day is ideal. Concerns have been expressed that reducing sodium intake too low could lead to maladaptive sympathetic and RAAS responses and some of this concern comes from studies where sodium/volume depletion diets were used (as low as 230 mg/d sodium). PURPOSE To assess the sympathetic and RAAS responses of reducing sodium intake from the widely recommended 2300 mg/d to 0.20). Reducing sodium intake to 1000 mg/d increased plasma renin activity (1.5±0.3 vs. 2.2±0.4ng/mL/hr, P0.20), burst incidence (23.9±2.3 vs. 21.8±3.0 bursts/100 heart beats, P>0.20), or plasma norepinephrine concentrations (237±46 vs. 173±57 pg/mL, P>0.20). CONCLUSION These preliminary data suggest that reducing sodium from 2300 mg/d to 1000 mg/d stimulates RAAS but does not alter resting MSNA or catecholamines in healthy young adults. These findings suggest that diets as low as 1000 mg Na + /day can be consumed without stimulating the sympathetic nervous system. Support or Funding Information Supported by NIH Grant 1R01HL128388 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The Effects of Aerobic Fitness on Blood Pressure Reactivity During Controlled Low and High Sodium Diets

Purpose Increased blood pressure (BP) reactivity to acute exercise is predictive of future cardiovascular events. Excess dietary sodium exaggerates centrally‐mediated BP reactivity in salt‐resistant rodents. Regular exercise attenuates BP reactivity in rodents, presumably through increased fitness. However, the influence of dietary sodium on BP reactivity has not been investigated in humans. Furthermore, the interaction between dietary sodium and aerobic fitness on neurovascular regulation has not been investigated in humans. Therefore, we sought to test the hypotheses that 1) high dietary sodium exaggerates BP reactivity to acute hand‐grip exercise in humans; and 2) high aerobic fitness modulates high dietary sodium‐induced alterations in BP reactivity in healthy, young adults. Methods Thirteen healthy, young adults (8F/5M, age: 28±2 yrs, BMI: 24.1±0.9 kg/m 2 ; BP: 109±2/63±2mmHg; Mean±SEM) completed randomized, controlled 10‐day diets of high (7 g/day) and low (1 g/day) dietary sodium separated by ≥ four weeks. Beat‐to‐beat laboratory BP was measured via photoplethysmography on day 10 of both diets. BP reactivity was assessed as ΔBP (mmHg) during the final minute of a two‐minute static hand‐grip exercise trial at 40% maximal voluntary contraction compared to a preceding 10‐minute baseline. Aerobic fitness was assessed as VO 2Peak using a progressive‐intensity treadmill exercise protocol and indirect calorimetry. Differences in high‐ vs. low‐sodium BP reactivity (high – low sodium ΔBP reactivity; using the final minute of hand‐grip exercise BP data) was compared by splitting the participants into two categories of fitness (low vs. high) and completing an independent‐samples t‐test. Low aerobic fitness was defined as a relative VO 2Peak of ≤40 ml*kg−1*min −1 . Using this criterion, seven participants were classified as having “low aerobic fitness” and six were classified as having “high aerobic fitness”. Twenty four‐hour urinary sodium excretion was measured to ensure dietary compliance. Results Urinary Na + excretion increased on the high‐ vs. low‐sodium diet (259.7±18.4 vs. 33.2±6.6 mmol/24 hours; p 0.05). The high sodium diet augmented systolic (24.2±2.6 vs. 17.4±3.1; p = 0.03), but not diastolic (18.7±1.8 vs. 15.5±2.5; p = 0.19) BP reactivity during acute hand‐grip exercise in all participants. There was a trend (p = 0.06) for the low aerobic fitness participants to demonstrate greater Δ systolic BP reactivity compared to the high aerobic fitness participants (11.5±4.3 vs. 1.1±2.4 ΔBP reactivity). However, there was not a statistically significant difference ( p = 0.32) between the low and high aerobic fitness participants regarding Δ diastolic BP reactivity between diets (5.5±3.5 vs. 0.7±2.9 ΔBP reactivity). Conclusion These preliminary data suggest that 1) high dietary sodium exaggerates systolic BP reactivity to acute hand‐grip exercise in humans; and 2) high aerobic fitness may modulate some of the neurovascular effects of high dietary sodium in healthy young adults, although more data is needed. Support or Funding Information NIH Grant 1RO1HL128388 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

A New Pathway of Transcription‐Coupled Repair

Transcription‐coupled repair (TCR) of damaged DNA in E. coli can involve several different pathways. We propose that one pathway entails release of RNAP at the site of DNA damage without dissociation of the RNA:DNA hybrid. The RNA can serve as a primer for PolI, which initiates the repair process. We are investigating how TCR repairs nalidixic acid‐induced DNA damage. Nalidixic acid, like Etoposide generates a DNA double‐strand break bearing a TopA adduct at each 5′ DNA end. We show that efficient repair requires two transcription factors. The first DksA, is a coiled‐coil protein that inserts into the RNAP secondary channel and, in concert with ppGpp, regulates the activity of certain promoters. The role of DksA in transcription elongation is undefined, although it is known to suppresses replisome – RNAP clashes. DksA plays a direct role in repair, rather than blocking access of GreA to RNAP. Thus, a Δ dksA mutation is not suppressed by a greA deletion. Recent work from the Murakami laboratory reveals that DksA binding distorts the structure of RNAP, opening the pincers, thus potentially reducing the processivity of transcription elongation. DksA in association with ppGpp, however, does not affect RNAP structure. We find that repair is more efficient in strains carrying an RNAP mutation that reduces ppGpp binding to site 2, the location of DksA insertion. We have isolated a suppressor of Δ dksA based on resistance to nalidixic acid. The mutation, rpoBQ148H , phenocopies RNAP bound to DksA in the absence of ppGpp, i.e. it opens the pincers and may favor release of elongating RNAP. These findings suggest that DksA may act as a transcription termination factor that can release RNAP without removing the RNA:DNA hybrid. The second, transcription termination protein Rho, is an RNA‐dependent DNA:RNA helicase. The defects in TCR of rho and dksA mutations are additive, suggesting that they act on different substrates. Whereas Rho is thought to release RNAP by unwinding the RNA:DNA helix, rho mutants that terminate transcription without releasing RNA have been isolated. Another reaction that entails elongation of an RNA:DNA hybrid by PolI is constitutive stable DNA replication (cSDR). In cSDR, elongation of an R‐loop by PolI creates a new origin of replication. We find that cSDR is dependent on DksA. Presumably the mechanisms of nalidixic acid DNA damage and cSDR both involve allowing access of PolI to the 3′ end of an RNA:DNA hybrid. Support or Funding Information GM037219 GM037219 NIH: GM037219 NIH: GM037219 GM037219 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Paraoxonase 1 Q192R genotype and activity affect homocysteine thiolactone levels in humans

Genetic or nutritional deficiencies in 1 carbon and homocysteine (Hcy) metabolism elevate Hcy‐ thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy‐thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio‐protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy‐thiolactone in vitro. However, whether Hcy‐ thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy‐thiolactone levels in humans was unknown. Here we show that the PON1–192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy‐thiolactone levels, normalized to creatinine. Carriers of the PON1–192R allele had significantly lowerHcy‐thiolactone/creatinine levels than individuals carrying the PON1–192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy‐ thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy‐ thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy‐thiolactone in humans and that the in terindividual variations in PON1 genotype/activity can modulate the pathology of hyperhomocysteinemia.—Perla‐Kaján, J., Borowczyk, K., Glowacki, R., Nygard, O., Jakubowski, H. Paraoxonase 1 Q192R genotype and activity affect homocysteine thiolactone levels in humans. 32, 6019–6024 (2018). www.fasebj.org