An economic analysis of immediate delivery and expectant monitoring in women with hypertensive disorders of pregnancy, between 34 and 37 weeks of gestation (HYPITAT-II).

OBJECTIVE: To assess the economic consequences of immediate delivery compared with expectant monitoring in women with preterm non-severe hypertensive disorders of pregnancy. DESIGN: A cost-effectiveness analysis alongside a randomised controlled trial (HYPITAT-II). SETTING: Obstetric departments of seven academic hospitals and 44 non-academic hospitals in the Netherlands. POPULATION: Women diagnosed with non-severe hypertensive disorders of pregnancy between 340/7 and 370/7  weeks of gestation, randomly allocated to either immediate delivery or expectant monitoring. METHODS: A trial-based cost-effectiveness analysis was performed from a healthcare perspective until final maternal and neonatal discharge. MAIN OUTCOME MEASURES: Health outcomes were expressed as the prevalence of respiratory distress syndrome, defined as the need for supplemental oxygen for >24 hours combined with radiographic findings typical for respiratory distress syndrome. Costs were estimated from a healthcare perspective until maternal and neonatal discharge. RESULTS: The average costs of immediate delivery (n = 352) were €10 245 versus €9563 for expectant monitoring (n = 351), with an average difference of €682 (95% confidence interval, 95% CI -€618 to €2126). This 7% difference predominantly originated from the neonatal admissions, which were €5672 in the immediate delivery arm and €3929 in the expectant monitoring arm. CONCLUSION: In women with mild hypertensive disorders between 340/7 and 370/7  weeks of gestation, immediate delivery is more costly than expectant monitoring as a result of differences in neonatal admissions. These findings support expectant monitoring, as the clinical outcomes of the trial demonstrated that expectant monitoring reduced respiratory distress syndrome for a slightly increased risk of maternal complications. TWEETABLE ABSTRACT: Expectant management in preterm hypertensive disorders is less costly compared with immediate delivery.

Dairy product consumption, dietary nutrient and energy density and associations with obesity in Australian adolescents.

BACKGROUND: Dairy intake is likely to influence dietary energy density (ED) and nutrient density (ND), which are factors representing aspects of dietary quality. Although evidence suggests dairy intake is unlikely to contribute to obesity, intake tends to decrease over adolescence, potentially as a result of concerns around weight gain. We examined associations between dairy intake, ED and ND, and investigated relationships with obesity in adolescents. METHODS: The present study comprised a cross-sectional study of 1613 14-year-olds in the Western Australian Pregnancy Cohort (Raine) Study. Adolescents completed a 212-item food frequency questionnaire. Nutrient Rich Food index 9.3 (NRF9.3) was used to estimate ND. Age-specific body mass index (BMI) and waist-height cut-offs were used to categorise obesity risk. RESULTS: Mean (SD) dairy intake was: 2.62 (1.51) servings daily; ED was 4.53 (0.83) (food and beverage) and 6.28 (1.33) (food only); ND was 373 (109). Dairy intake was inversely associated with ED and positively associated with ND. The odds of being overweight (as assessed by BMI) increased by 1.24 (95% confidence interval = 1.09-1.42) with each 100-point increase in ND, after adjustment for potential confounders and energy intake. ED measures and dairy intake were inversely associated with obesity after adjustment for confounders; associations became nonsignificant after energy adjustment. CONCLUSIONS: The NRF9.3 was originally designed to assess foods, not diets. Further research in other cohorts to determine whether similar findings exist, or investigations into alternate measures of dietary ND, may prove useful. Our findings may be the result of factors such as an excess consumption of refined but fortified foods. Although higher dairy intakes were associated with higher ND, intakes were not associated with higher obesity risk.

Transfusion policy after severe postpartum haemorrhage: a randomised non-inferiority trial.

OBJECTIVE: To assess the effect of red blood cell (RBC) transfusion on quality of life in acutely anaemic women after postpartum haemorrhage. DESIGN: Randomised non-inferiority trial. SETTING: Thirty-seven Dutch university and general hospitals. POPULATION: Women with acute anaemia (haemoglobin 4.8-7.9 g/dl [3.0-4.9 mmol/l] 12-24 hours postpartum) without severe anaemic symptoms or severe comorbidities. METHODS: Women were allocated to RBC transfusion or non-intervention. MAIN OUTCOME MEASURES: Primary outcome was physical fatigue 3 days postpartum (Multidimensional Fatigue Inventory, scale 4-20; 20 represents maximal fatigue). Non-inferiority was demonstrated if the physical fatigue difference between study arms was maximal 1.3. Secondary outcomes were health-related quality of life and physical complications. Health-related quality of life questionnaires were completed at five time-points until 6 weeks postpartum. RESULTS: In all, 521 women were randomised to non-intervention (n = 262) or RBC transfusion (n = 259). Mean physical fatigue score at day 3 postpartum, adjusted for baseline and mode of delivery, was 0.8 lower in the RBC transfusion arm (95% confidence interval: 0.1-1.5, P = 0.02) and at 1 week postpartum was 1.06 lower (95% confidence interval: 0.3-1.8, P = 0.01). A median of two RBC units was transfused in the RBC transfusion arm. In the non-intervention arm, 33 women received RBC transfusion, mainly because of anaemic symptoms. Physical complications were comparable. CONCLUSIONS: Statistically, non-inferiority could not be demonstrated as the confidence interval crossed the non-inferiority boundary. Nevertheless, with only a small difference in physical fatigue and no differences in secondary outcomes, implementation of restrictive management seems clinically justified.

The JAK1/2 inhibitor ruxolitinib in patients with COVID-19 triggered hyperinflammation: the RuxCoFlam trial.

Dysregulated hyperinflammatory response is key in the pathogenesis in patients with severe COVID-19 leading to acute respiratory distress syndrome and multiorgan failure. Whilst immunosuppression has been proven to be effective, potential biological targets and optimal timing of treatment are still conflicting. We sought to evaluate efficacy and safety of the Janus Kinase 1/2 inhibitor ruxolitinib, employing the previously developed COVID-19 Inflammation Score (CIS) in a prospective multicenter open label phase II trial (NCT04338958). Primary objective was reversal of hyperinflammation (CIS reduction of ≥25% at day 7 in ≥20% of patients). In 184 patients with a CIS of ≥10 (median 12) ruxolitinib was commenced at an initial dose of 10 mg twice daily and applied over a median of 14 days (range, 2-31). On day 7, median CIS declined to 6 (range, 1-13); 71% of patients (CI 64-77%) achieved a ≥25% CIS reduction accompanied by a reduction of markers of inflammation. Median cumulative dose was 272.5 mg/d. Treatment was well tolerated without any grade 3-5 adverse events related to ruxolitinib. Forty-four patients (23.9%) died, all without reported association to study drug. In conclusion, ruxolitinib proved to be safe and effective in a cohort of COVID-19 patients with defined hyperinflammation.

[Interdisciplinary COVID board for patients with SARS-CoV-2-triggered hyperferritinemic Inflammation]. / Interdisziplinäres COVID-Board bei SARS-CoV-2-getriggerter hyperferritinämischer Inflammation.

BACKGROUND: Patients with severe COVID-19 develop hyperferritinemic inflammation, a rare sepsis-like immune dysregulation syndrome. METHODS: Stratified treatment decisions in a cross-location telemedical interdisciplinary case conference were assessed in this retrospective cohort study. A standardized treatment algorithm including continuous positive airway pressure and noninvasive ventilation was implemented. A locally developed COVID inflammation score (CIS) defined patients at risk for severe disease. Patients with life-threatening inflammation were offered off-label treatment with the immune modulator ruxolitinib. RESULTS: Between 4 March 2020 and 26 June 2020 COVID-19 patients (n = 196) were treated. Median patient age (70 years) and comorbidity were high in interstudy comparison. Mortality in all patients was 17.3%. However, advance care planning statements and physician directives limited treatment intensity in 50% of the deceased patients. CIS monitoring of ruxolitinib-treated high-risk patients (n = 20) on days 5, 7, and15 resulted in suppression of inflammation by 42% (15-70), 54% (15-77) and 60% (15-80). Here, mortality was 20% (4/20). Adjusted for patients with a maximum care directive including ICU, total mortality was 8.7% (17/196). CONCLUSION: Severe COVID-19 pneumonia with hyperferritinemic inflammation is related to macrophage activation syndrome-like sepsis. An interdisciplinary intensive care teleconference as a quality tool for ICUs is proposed to detect patients with rare sepsis-like syndromes.

The Janus kinase 1/2 inhibitor ruxolitinib in COVID-19 with severe systemic hyperinflammation.

A subgroup of patients with severe COVID-19 suffers from progression to acute respiratory distress syndrome and multiorgan failure. These patients present with progressive hyperinflammation governed by proinflammatory cytokines. An interdisciplinary COVID-19 work flow was established to detect patients with imminent or full blown hyperinflammation. Using a newly developed COVID-19 Inflammation Score (CIS), patients were prospectively stratified for targeted inhibition of cytokine signalling by the Janus Kinase 1/2 inhibitor ruxolitinib (Rux). Patients were treated with efficacy/toxicity guided step up dosing up to 14 days. Retrospective analysis of CIS reduction and clinical outcome was performed. Out of 105 patients treated between March 30th and April 15th, 2020, 14 patients with a CIS ≥ 10 out of 16 points received Rux over a median of 9 days with a median cumulative dose of 135 mg. A total of 12/14 patients achieved significant reduction of CIS by ≥25% on day 7 with sustained clinical improvement in 11/14 patients without short term red flag warnings of Rux-induced toxicity. Rux treatment for COVID-19 in patients with hyperinflammation is shown to be safe with signals of efficacy in this pilot case series for CRS-intervention to prevent or overcome multiorgan failure. A multicenter phase-II clinical trial has been initiated (NCT04338958).

Ketonuria is not associated with hyperemesis gravidarum disease severity.

OBJECTIVE: To assess the association between ketonuria and hyperemesis gravidarum (HG) disease severity. STUDY DESIGN: We included pregnant women hospitalised for HG who participated in the Maternal and Offspring outcomes after Treatment of HyperEmesis by Refeeding (MOTHER) trial and women who were eligible, chose not to be randomised and agreed to participate in the observational cohort. Between October 2013 and March 2016, in 19 hospitals in the Netherlands, women hospitalised for HG were approached for study participation. The presence of ketonuria was not required for study entry. Ketonuria was measured at hospital admission with a dipstick, which distinguishes 5 categories: negative and 1+ through 4 + . The outcome measures were multiple measures of HG disease severity at different time points: 1) At hospital admission (study entry): severity of nausea and vomiting, quality of life and weight change compared to pre-pregnancy weight, 2) One week after hospital admission: severity of nausea and vomiting, quality of life and weight change compared to admission, 3) Duration of index hospital admission and readmission for HG at any time point RESULTS: 215 women where included. Ketonuria was not associated with severity of nausea and vomiting, quality of life or weight loss at hospital admission, nor was the degree of ketonuria at admission associated with any of the outcomes 1 week after hospital admission. The degree of ketonuria was also not associated with the number of readmissions. However, women with a higher degree of ketonuria had a statistically significant longer duration of hospital stay (per 1+ ketonuria, difference: 0.27 days, 95 % CI: 0.05 to 0.48). CONCLUSIONS: There was no association between the degree of ketonuria at admission and severity of symptoms, quality of life, maternal weight loss, or number of readmissions, suggesting that ketonuria provides no information about disease severity or disease course. Despite this, women with a higher degree of ketonuria at admission were hospitalised for longer. This could suggest that health care professionals base length of hospital stay on the degree of ketonuria. Based on the lack of association between ketonuria and disease severity, we suggest it has no additional value in the clinical management of HG.

Varying rate of RNA chain elongation during rrn transcription in Escherichia coli.

The value of the rRNA chain elongation rate in bacteria is an important physiological parameter, as it affects not only the rRNA promoter activity but also the free-RNA polymerase concentration and thereby the transcription of all genes. On average, rRNA chains elongate at a rate of 80 to 90 nucleotides (nt) per s, and the transcription of an entire rrn operon takes about 60 s (at 37 degrees C). Here we have analyzed a reported distribution obtained from electron micrographs of RNA polymerase molecules along rrn operons in E. coli growing at 2.5 doublings per hour (S. Quan, N. Zhang, S. French, and C. L. Squires, J. Bacteriol. 187:1632-1638, 2005). The distribution exhibits two peaks of higher polymerase density centered within the 16S and 23S rRNA genes. An evaluation of this distribution indicates that RNA polymerase transcribes the 5' leader region at speeds up to or greater than 250 nt/s. Once past the leader, transcription slows down to about 65 nt/s within the 16S gene, speeds up in the spacer region between the 16S and 23S genes, slows again to about 65 nt/s in the 23S region, and finally speeds up to a rate greater than 400 nt/s near the end of the operon. We suggest that the slowing of transcript elongation in the 16S and 23S sections is the result of transcriptional pauses, possibly caused by temporary interactions of the RNA polymerase with secondary structures in the nascent rRNA.

Compliance in sleep apnoea therapy: influence of home care support and pressure mode.

Continuous positive airway pressure (CPAP) is an effective treatment for obstructive sleep apnoea syndrome (OSAS) but therapy adherence is often low. The hypothesis that CPAP-adherence and clinical outcomes can be improved by either using an autoadjusting-CPAP (APAP) device or an intensive support was tested. A controlled parallel group study was performed with 100 newly diagnosed OSAS patients, randomised into 4 groups (n = 25 each): standard or intensive support plus either APAP or CPAP. Intensive support included education and monthly home visits for 6 months. Clinical outcome was monitored by polysomnography at CPAP initiation and, after 3 and 9 months, compliance data were downloaded from the CPAP devices. After 9 months, intensively supported patients returned for follow-up in 88 versus 68% in the standard-support-group. Daily usage (mean+/-sem 5.7+/-0.2 for intensive support versus 4.6+/-0.4 h for standard support), percentage of days used (80.4+/-2.8 versus 57.0+/-5.9%) and proportion of individual sleep time (80.6+/-3.2 versus 64.9+/-6.2%) were also higher. There was no significant difference between APAP or CPAP, (daily usage 5.2+/-0.4 versus 5.1+/-0.3 h, percentage of days 67.9+/-5.0 versus 69.2+/-4.9%, proportion of sleep time 72.5+/-5.0% versus 72.1+/-5.2%, for APAP and CPAP) but retention rate was higher with CPAP. In summary, intensive support after continuous positive airway pressure initiation, rather than the application of autoadjusting-continuous positive airway pressure, increased therapy adherence.

Feedback control of ribosome function in Escherichia coli.

We have previously proposed that the rate of ribosome function during balanced growth in E. coli, expressed as the rate of peptide chain elongation, is adjusted by a feedback mechanism: whenever that rate is submaximal (i.e. below 22 amino acid residues polymerized per active ribosome at 37 degrees C), the feedback signal ppGpp is generated by an activation of the ppGpp synthetase expressed from the spoT gene. The accumulation of ppGpp reduces the synthesis of additional ribosomes and thereby reduces the consumption of amino acids which, in turn, allows the remaining ribosomes to function at a higher rate. Here we have described with supporting evidence the proposed feedback loop in greater detail and provided a mathematical analysis which predicts that the SpoT ppGpp synthetase activity should be highest when the ribosomes function at their half-maximal rate. This prediction is consistent with reported observations and is independent of the particular (unknown) mechanism by which the rate of translation controls the ppGpp synthetase activity of SpoT.

Large neutral amino acids block phenylalanine transport into brain tissue in patients with phenylketonuria.

Large neutral amino acids (LNAAs), including phenylalanine (Phe), compete for transport across the blood-brain barrier (BBB) via the L-type amino acid carrier. Accordingly, elevated plasma Phe impairs brain uptake of other LNAAs in patients with phenylketonuria (PKU). Direct effects of elevated brain Phe and depleted LNAAs are probably major causes for disturbed brain development and function in PKU. Competition for the carrier might conversely be put to use to lower Phe influx when the plasma concentrations of all other LNAAs are increased. This hypothesis was tested by measuring brain Phe in patients with PKU by quantitative 1H magnetic resonance spectroscopy during an oral Phe challenge with and without additional supplementation with all other LNAAs. Baseline plasma Phe was approximately 1,000 micromol/l and brain Phe was approximately 250 micromol/l in both series. Without LNAA supplementation, brain Phe increased to approximately 400 micromol/l after the oral Phe load. Electroencephalogram (EEG) spectral analysis revealed acutely disturbed brain activity. With concurrent LNAA supplementation, Phe influx was completely blocked and there was no slowing of EEG activity. These results are relevant for further characterization of the LNAA carrier and of the pathophysiology underlying brain dysfunction in PKU and for treatment of patients with PKU, as brain function might be improved by continued LNAA supplementation.

[Pulmonary hypertension due to chronic lung disease: Recommendations of the Cologne Consensus Conference 2016]. / Pulmonale Hypertonie bei Lungenkrankheiten: Empfehlungen der Kölner Konsensus-Konferenz 2016.

The 2015 European Guidelines on Pulmonary Hypertension did not cover only pulmonary arterial hypertension (PAH) but also some aspects of pulmonary hypertension (PH) associated with chronic lung disease. The European Guidelines point out that the drugs currently used to treat patients with PAH (prostanoids, endothelin receptor antagonists, phosphodiesterase-5 inhibitors, sGC stimulators) have not been sufficiently investigated in other forms of PH. Therefore, the European Guidelines do not recommend the use of these drugs in patients with chronic lung disease and PH. This recommendation, however, is not always in agreement with medical ethics as physicians feel sometimes inclined to treat other form of PH which may affect quality of life and survival of these patients in a similar manner. To this end, it is crucial to consider the severity of both PH and the underlying lung disease. In June 2016, a Consensus Conference organized by the PH working groups of the German Society of Cardiology (DGK), the German Society of Respiratory Medicine (DGP) and the German Society of Pediatric Cardiology (DGPK) was held in Cologne, Germany, to discuss open and controversial issues surrounding the practical implementation of the European Guidelines. Several working groups were initiated, one of which was dedicated to the diagnosis and treatment of PH in patients with chronic lung disease. The recommendations of this working group are summarized in the present paper.

Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing.

A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp-synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis.

Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia coli.

The DNA-bending protein Fis of Escherichia coli is required for efficient initiation of chromosome replication and for activation of the P1 promoters of stable RNA (rRNA and tRNA) genes. Using fis+ and fis- bacterial strains, we have determined ribosome synthesis and activity, rRNA gene dosage, and transcript initiation rates at the rrnB P1 and P2 promoters. Different growth media were used to achieve growth rates between 0.7 and 2.7 doublings/hour for the fis+ strain, and between 0.7 and 2.2 doublings/hour for the fis- strain. In minimal media, the Fis deficiency only reduced the DNA and rrn gene concentration (DNA, oriC copies and rrn genes per protein); in amino acid-supplemented media, it also reduced rRNA synthesis rates per protein. Under all conditions, the ribosome activity (protein synthesis rate/ribosome) remained unchanged by the absence of Fis. In the presence of Fis, the absolute activities of the isolated rrnB P1 and P2 promoters increased from 2 to 88 and 10 to 50 initiations/minute, respectively, with increasing growth-rate. In the absence of Fis, these activities increased from 3 to 70 and 10 to 80 initiations/minute, respectively. Relative to the isolated rrnB P2 promoter, the strength of the rrnB P1 promoter was found to increase with increasing growth rate tenfold (from 0.17 to 1.7) in the presence, but only fivefold (from 0.17 to 0.85) in the absence of Fis. An evaluation of the data leads us to propose estimates of kcat and relative KM values for the two rRNA promoters, and relative values for free RNA polymerase concentrations during growth in different media. The analysis suggests that the reduced strength of P1 promoters of stable RNA genes in the absence of Fis, together with the reduced rrn gene concentration, increases the concentration of free RNA polymerase. In addition, the lower rrn P1 promoter activity in the absence of Fis reduces the probability that the downstream P2 promoter is blocked ("occluded") by a transcription elongation complex originating at the P1 promoter. The increased polymerase concentration and reduced P2 promoter occlusion both help to compensate for the Fis-deficiency, but during growth in rich media when the demand for ribosomes is high, this compensation is insufficient for fis- bacteria to achieve the wild-type level of ribosome synthesis and growth.

Control of spoT-dependent ppGpp synthesis and degradation in Escherichia coli.

Escherichia coli has two ppGpp synthetases, PSI and PSII, encoded by the relA and spoT genes. The spoT gene also encodes a ppGpp hydrolase. During exponential growth and under various starvation conditions, the level of ppGpp depends on the balance of ppGpp synthetic and degradative activities of spoT gene products. To find out how these two activities respond to different physiological conditions and to learn about the signals involved in these responses, rates of ppGpp synthesis and degradation were determined in an E. coli B/r delta relA strain during: (1) multiple amino acid deprivation after a nutritional shift-down from glucose amino acids to glucose minimal medium; (2) carbon source starvation after a "glucose runout"; (3) energy starvation by treatment with sodium azide. To each of these conditions, bacteria responded with a similar gradual accumulation of ppGpp, occurring over a period of 20 to 40 minutes, from the basal level of 4 and 24 pmol/OD460 in glucose amino acids and glucose minimal medium, respectively, to about 100 pmol ppGpp/OD460 unit of culture mass. After multiple amino acid deprivation and during azide treatment, the rate of ppGpp synthesis increased and the rate of ppGpp degradation decreased, but in different proportions by the two kinds of treatment. After glucose runout, both ppGpp synthesis and degradation immediately decreased, but the rate of degradation was reduced more, which caused the accumulation of ppGpp despite its reduced synthesis. ppGpp synthesis required continuous protein synthesis, but ppGpp hydrolysis and its control did not: the rate of ppGpp degradation could be instantly up or down-regulated in response to changes in exogenous amino acid or glucose levels in the absence of protein synthesis. The results suggest that PSII is unstable with an average functional lifetime of 40 seconds or less, and that its activity is generated during or shortly after spoT mRNA translation in response to the availability of amino acids. This regulation is responsible for the growth medium-dependent changes in basal levels of ppGpp. ppGpp hydrolysis is controlled, i.e. inhibited, mainly during conditions of physiological stress, such as multiple amino acid deprivation or energy deprivation. This inhibition can be correlated with an inferred accumulation of uncharged tRNA. Since previous reports have indicated that uncharged tRNA inhibits purified SpoT hydrolase in vitro, it is proposed that ppGpp hydrolase activity is, indeed, controlled by the concentration of uncharged tRNA in the cell. Finally, it was found that neither relC, transcriptional regulation of spoT, nor different translation starts of spoT mRNA are directly involved in the environmental control of SpoT hydrolase and PSII activities.

Decay of rplN and lacZ mRNA in Escherichia coli.

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of beta-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5' and 3'-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay.

Activities of constitutive promoters in Escherichia coli.

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains.

Disease patterns and incidence of immune-mediated disease in insured Swedish Nova Scotia Duck Tolling Retrievers.

In this study, morbidity in insured Nova Scotia Duck Tolling Retriever (NSDTR) dogs from Sweden was investigated and compared with all other breeds and other retriever breeds. In addition to describing common morbidities in NSDTRs, the hypotheses that NSDTRs are predisposed to lymphoma, immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis-arteritis (SRMA) were tested. Included in the study were 445,336 dogs; of which, 2890 were NSDTRs that had been covered by veterinary insurance from the Agria Insurance Company (Stockholm, Sweden) at some point during the years 1995-2006. Incidences of various health problems were calculated using the number of veterinary visits as the numerator and the exact time at risk as the denominator. Overall, morbidity was higher in NSDTRs compared with all other breeds, but similar compared with other retriever breeds. The most common causes of veterinary visits in NSDTRs were injuries, gastrointestinal disease and locomotor disorders, with NSDTRs at increased risk of these compared with all other breeds. The incidences for IMRD, SRMA and lymphoma were significantly higher in NSDTRs than in all other dog breeds and all other retriever breeds. The study describes morbidity in NSDTRs, and identifies several disorders to which the breed is predisposed.

Detection of mannose-binding proteins on mouse lymphocytes.

D-Mannose influences both the allogeneic stimulation of mouse spleen cells and the induction of cytotoxic T-lymphocytes. At low concentrations (10(-3) -4 x 10(-3) M), D-mannose increases [3H]thymidine incorporation and induction of cytotoxic cells. Higher concentrations lead to an inhibition of the allogeneic response. To test the involvement of mannose receptors, unstimulated and allogeneically stimulated spleen cells were lysed and the proteins electrophoretically separated. After blotting, the D-mannose-binding structures were identified by means of a biotinylated mannose-neoglycoprotein and avidin peroxidase. Two mannose-binding proteins of unstimulated mouse spleen lymphocytes with a molecular mass of 29 and 31 kDa were detected. In vitro allogeneically stimulated spleen lymphocytes show three other mannose-binding proteins with a molecular mass of 53, 65, and 76 kDa.