Do improvements in clinical practice guidelines alter pregnancy outcomes in asthmatic women? A single-center retrospective cohort study.

OBJECTIVE: Asthma occurs in ∼17% of Australian pregnancies and is associated with adverse perinatal outcomes, which worsen with poor asthma control. Consequently, the South Australian 'Asthma in Pregnancy' perinatal guidelines were revised in 2012 to address management according to severity. This study investigated if these revised guidelines reduced the impact of maternal asthma on risks of adverse perinatal outcomes before (Epoch 1, 2006-2011) and after the revision (Epoch 2, 2013-2018). METHODS: Routinely collected perinatal and neonatal datasets from the Women's and Children's Hospital (Adelaide, Australia) were linked. Maternal asthma (prevalence:7.5%) was defined as asthma medication use or symptoms described to midwives. In imputation (n = 59131) and complete case datasets (n = 49594), analyses were conducted by inverse proportional weighting and multivariate logistic regression, accounting for confounders. RESULTS: Overall, maternal asthma was associated with increased risks of any antenatal corticosteroid treatment for threatened preterm birth (aOR 1.319, 95% CI 1.078-1.614), any Cesarean section (aOR 1.196, 95% CI 1.059-1.351), Cesarean section without labor (aOR 1.241, 95% CI 1.067-1.444), intrauterine growth restriction (IUGR, aOR 1.285, 95% CI 1.026-1.61), and small for gestational age (aOR 1.324, 95% CI 1.136-1.542). After guideline revision, asthma-associated risks of any Cesarean section (p < 0.001), any antenatal corticosteroids (p = 0.041), and small for gestational age (p = 0.050), but not IUGR and Cesarean section without labor, were reduced. CONCLUSIONS: Clinical practice guidelines based on the latest evidence do not guarantee clinical efficacy. Since adverse perinatal outcomes did not all improve, this work highlights the need to evaluate the ongoing impact of guidelines on clinical outcomes.

Reduction of biological response modifiers in the supernatant of washed paediatric red blood cells.

BACKGROUND: Washing of red blood cells (RBC) can reduce unwanted biological response modifiers (BRMs) that can mediate transfusion complications in infants. The aim of this study was to examine the in vitro quality and the changes in BRMs following washing in paediatric RBC units. MATERIALS AND METHODS: A pool and split design was used to prepare RBC (either 1 or 4 days old; n = 26 pairs). One unit was washed with 0·9% saline by centrifugation and then resuspended in SAG-M, while the other remained unwashed. Each RBC unit was divided to produce four units of paediatric-sized components. Samples were taken after 3 h and subsequently on days 1, 2, 7 and 14 post-wash. RESULTS: Washing of RBC resulted in some red cell loss, with a minor increase in haemolysis. Washing effectively reduced supernatant potassium and IgA, as well as cytokines and complement proteins. RBC microparticles were significantly reduced in RBC washed at 1, but not 4 days post-collection. Incubation with supernatant from unwashed but not washed RBC led to endothelial cell activation, with increased cell surface expression of CD62E (E-selectin) and CD106 (VCAM). CONCLUSION: Although washing affected some aspects of the in vitro quality of RBC, it effectively reduced the concentration and activity of BRMs in the supernatant of RBC. Such a reduction may be clinically beneficial in selected patient groups.

Redox-mediated substrate recognition by Sdp1 defines a new group of tyrosine phosphatases.

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.

Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.

Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication.

Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.

Intracellular membrane fusion is crucial for the biogenesis and maintenance of cellular compartments, for vesicular traffic between them, and for exo- and endocytosis. Parts of the molecular machinery underlying this process have been identified, but most of these components operate in mutual recognition of the membranes. Here it is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion. PP1 was also identified in a complex that contained calmodulin, the second known factor implicated in the regulation of bilayer mixing. The PP1-calmodulin complex was required at multiple sites of intracellular trafficking; hence, PP1 may be a general factor controlling membrane bilayer mixing.

The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.

In higher eukaryotes, the activity and specificity of the type 1 protein serine-threonine phosphatase (PP1) catalytic subunit is thought to be controlled by its association with a number of regulatory or targeting subunits. Here we describe the characterization of a gene encoding one such potential polypeptide in the yeast Saccharomyces cerevisiae. The gene which we have isolated (termed SDS22) encodes a product with a high degree of sequence identity to the fission yeast sds22 protein, a known regulator of the mitotic function of PP1 in Schizosaccharomyces pombe. Using two different criteria, we have demonstrated that Sds22p and the catalytic subunit of PP1 (Glc7p) interact in yeast cells. We have also generated a temperature-sensitive allele of GLC7 (glc7-12) which causes a block to the completion of mitosis at the restrictive temperature. Additional copies of SDS22 lead to allele-specific suppression of the glc7-12 mutant, strongly suggesting that the interaction between the two proteins is of functional significance. Sds22p is therefore likely to be the second example of a PP1 regulatory subunit identified in S. cerevisiae.

Two Saccharomyces cerevisiae genes which control sensitivity to G1 arrest induced by Kluyveromyces lactis toxin.

The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.

Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation.

The 110 kDa spindle pole body component, Spc110p, is an essential target of calmodulin in budding yeast. Cells with mutations which reduce calmodulin binding to Spc110p are unable to form a mitotic spindle and die. Here we show that these effects can be overcome either directly by increasing extracellular calcium or calmodulin expression, which reverse the primary spindle defect, or indirectly through increased extracellular osmolarity or high dosage of MID2 or SLG1/HCS77/WSC1 which preserve viability. We propose that overcoming a cell integrity defect associated with the mitotic arrest enables the defective spindle pole bodies to provide sufficient function for proliferation of a large proportion of mutant cells. Our findings demonstrate a role for calcium in the Spc110p-calmodulin interaction in vivo and have important general implications for the interpretation of genetic interactions involving cell integrity genes.

Turnover of rat liver malic enzyme during induction by protein deprivation.

The half-lives of hepatic malic enzyme and total liver soluble proteins were determined in protein-sufficient and protein-deficient rats after injection of tracer doses of radioactively labeled leucine. The results obtained in these experiments have demonstrated that the increased levels of malic enzyme obtained under conditions of severe protein restriction are due to elevated rates of synthesis of the enzyme protein, with no apparent change in the rate of its degradation.

Protein phosphatase 2Bw and protein phosphatase Z are Saccharomyces cerevisiae enzymes.

cDNAs encoding three protein phosphatases, termed PP2Bw (Da Cruz e Silva, E.F. and Cohen, P.T.W. (1989) Biochim. Biophys. Acta 1009, 293-296), PPZ1 and PPZ2 that have been isolated from a Clontech 'rabbit brain' library are shown to be Saccharomyces cerevisiae clones. PPZ1 and PPZ2 are two novel yeast phosphatases showing 93% amino acid sequence identity to one another. PPZ1 shows approx. 60% sequence identity to S. cerevisiae or mammalian PP1 and approx. 40% identity to S. cerevisiae or mammalian PP2A. These and other observations suggest that the two isoforms of PPZ have functions distinct from those of PP1.

A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro.

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.

Interaction of ribosomal proteins S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA from Escherichia coli.

The co-operative interaction of 30 S ribosomal subunit proteins S6, S8, S15 and S18 with 16 S ribosomal RNA from Escherichia coli was studied by (1) determining how the binding of each protein is influenced by the others and (2) characterizing a series of protein-rRNA fragment complexes. Whereas S8 and S15 are known to associate independently with the 16 S rRNA, binding of S18 depended upon S8 and S15, and binding of S6 was found to require S8, S15 and S18. Ribonucleoprotein (RNP) fragments were derived from the S8-, S8/S15- and S6/S8/S15/S18-16 S rRNA complexes by partial RNase hydrolysis and isolated by electrophoresis through Mg2+-containing polyacrylamide gels or by centrifugation through sucrose gradients. Identification of the proteins associated with each RNP by gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of S8, S8 + S15 and S6 + S8 + S15 + S18 in the corresponding fragment complexes. Analysis of the rRNA components of the RNP particles confirmed that S8 was bound to nucleotides 583 to 605 and 624 to 653, and that S8 and S15 were associated with nucleotides 583 to 605, 624 to 672 and 733 to 757. Proteins S6, S8, S15 and S18 were shown to protect nucleotides 563 to 605, 624 to 680, 702 to 770, 818 to 839 and 844 to 891, which span the entire central domain of the 16 S rRNA molecule (nucleotides 560 to 890). The binding site for each protein contains helical elements as well as single-stranded internal loops ranging in size from a single bulged nucleotide to 20 bases. Three terminal loops and one stem-loop structure within the central domain of the 16 S rRNA were not protected in the four-protein complex. Interestingly, bases within or very close to these unprotected regions have been shown to be accessible to chemical and enzymatic probes in 30 S subunits but not in 70 S ribosomes. Furthermore, nucleotides adjacent to one of the unprotected loops have been cross-linked to a region near the 3' end of 16 S rRNA. Our observations and those of others suggest that the bases in this domain that are not sequestered by interactions with S6, S8, S15 or S18 play a role involved in subunit association or in tertiary interactions between portions of the rRNA chain that are distant from one-another in the primary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.

Using a multicopy plasmid encoding the Escherichia coli rrnB ribosomal RNA operon and the techniques of in vitro site-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein-16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations of E. coli ribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis.

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37 degrees), cells dependent solely on pph22 alleles for PP2A function displayed a rapid arrest of proliferation. Ts pph22 mutant cells underwent lysis at 37 degrees, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37 degrees. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24 degrees to 37 degrees, Ts- pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37 degrees, many Ts- pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts- pph22 mutant cells also accumulated aberrant microtubule structures at 37 degrees, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts- pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.

Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin.

We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G(1) phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190. Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also confer K. lactis zymocin resistance. Since sit4Delta and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.

Association of early and late maternal smoking during pregnancy with offspring body mass index at 4 to 5 years of age.

The objective was to investigate the association between early and late maternal smoking during pregnancy on offspring body mass index (BMI). We undertook a retrospective cohort study using linked records from the Women's and Children's Health Network in South Australia. Among a cohort of women delivering a singleton, live-born infants between January 2000 and December 2005 (n=7658), 5961 reported not smoking during pregnancy, 297 reported quitting smoking during the first trimester of pregnancy, and 1400 reported continued smoking throughout pregnancy. Trained nurses measured the height and weight of the children at preschool visits in a state-wide surveillance programme. The main outcome measure was age- and sex-specific BMI z-score. At 4 to 5 years, mean (s.d.) BMI z-score was 0.40 (1.05), 0.60 (1.07) and 0.65 (1.18) in children of mothers who reported never smoking, quitting smoking and continued smoking during pregnancy, respectively. Compared with the group of non-smokers, both quitting smoking and continued smoking were associated with an increase in child BMI z-score of 0.15 (95% confidence interval: 0.01-0.29) and 0.21 (0.13-0.29), respectively. A significant dose-response relationship was also observed between the number of cigarettes smoked per day on average during the second half of pregnancy and the increase in offspring BMI z-score (P<0.001). In conclusion, any maternal smoking in pregnancy, even if mothers quit, is associated with an increase in offspring BMI at 4 to 5 years of age.

Early red cell transfusion favourably alters cerebral oxygen extraction in very preterm newborns.

BACKGROUND: Elevated cerebral fractional tissue oxygen extraction (cFTOE; ≥0.4) predicts early brain injury in very preterm infants. While blood transfusion increases oxygen-carrying capacity, its ability to improve cerebral oxygen kinetics in the immediate newborn period remains unknown. OBJECTIVE: To investigate the effect of red blood cell (RBC) transfusion in the first 24 h of life on cFTOE in infants ≤29 weeks gestation. METHODS: cFTOE was calculated from cerebral tissue oxygenation index (TOI) and cutaneous oximetry measured over a 30 min epoch before and after transfusion. Infants were dichotomised according to pre-transfusion cFTOE (low <0.4 vs high ≥0.4). RESULTS: 24 babies were included, 12 in each group. Pre- and post-transfusion Hb were similar between the groups. cFTOE significantly reduced after transfusion in the high but not low-extraction group (p<0.01). CONCLUSIONS: Early RBC transfusion favourably alters cerebral oxygen kinetics in infants with elevated cFTOE, showing potential for modification of the risk of hypoxic (brain) injury.

Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight.

INTRODUCTION: Administration of betamethasone to women at risk of preterm delivery is known to be associated with reduced fetal growth via alterations in placental function and possibly direct effects on the fetus. The placental glucocorticoid receptor (GR) is central to this response and recent evidence suggests there are numerous isoforms for GR in term placentae. In this study we have questioned whether GR isoform expression varies in preterm placentae in relation to betamethasone exposure, fetal sex and birthweight. METHODS: Preterm (24-36 completed weeks of gestation, n = 55) and term placentae (>37 completed weeks of gestation, n = 56) were collected at delivery. Placental GR expression was examined using Western Blot and analysed in relation to gestational age at delivery, fetal sex, birthweight and betamethasone exposure. Data was analysed using non-parametric tests. RESULTS: Eight known isoforms of the GR were detected in the preterm placenta and include GRα (94 kDa), GRß (91 kDa), GRα C (81 kDa) GR P (74 kDa) GR A (65 kDa), GRα D1-3 (50-55 kDa). Expression varied between preterm and term placentae with a greater expression of GRα C in preterm placentae relative to term placentae. The only sex differences in preterm placentae was that GRα D2 expression was higher in males than females. There were no alterations in preterm placental GR expression in association with betamethasone exposure. DISCUSSION: GRα C is the isoform involved in glucocorticoid induced apoptosis and suggests that its predominance in preterm placentae may contribute to the pathophysiology of preterm birth.

A dose response randomised controlled trial of docosahexaenoic acid (DHA) in preterm infants.

Thirty one infants born less than 30 weeks׳ gestational age were randomised to receive either 40 (n=11), 80 (n=9) or 120 (n=11) mg/kg/day of docosahexaenoic acid (DHA) respectively as an emulsion, via the feeding tube, commenced within 4 days of the first enteral feed. Twenty three infants were enroled in non-randomised reference groups; n=11 who had no supplementary DHA and n=12 who had maternal DHA supplementation. All levels of DHA in the emulsion were well tolerated with no effect on number of days of interrupted feeds or days to full enteral feeds. DHA levels in diets were directly related to blood DHA levels but were unrelated to arachidonic acid (AA) levels. All randomised groups and the maternal supplementation reference group prevented the drop in DHA levels at study end that was evident in infants not receiving supplementation. Australian New Zealand Clinical Trials Registry: ACTRN12610000382077.

Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli.

A series of new expression vectors (the pTTQ series) has been constructed for the regulated expression of genes in Escherichia coli. Based on the pUC plasmids, the pTTQ vectors contain a polylinker/lacZ alpha region flanked by the strong hybrid trp-lac (tac) promoter and the rrnB transcription terminator. Foreign genes can be inserted into the polylinker region of this expression cassette, to give either transcriptional or translational fusions within the lacZ alpha coding region. In most commonly used strains of E. coli, multiple copies of the lac operator titrate out the lac repressor. This phenomenon leads to significant expression from tac or lac promoters present on multicopy plasmids, even in the absence of inducers such as IPTG. To ensure maximal repression of the tac promoter on the pTTQ vectors in any host strain, the lacIQ allele of the lac repressor gene was added to the vectors. This makes them particularly useful for cloning genes when expression at high level is desired but is detrimental to cell growth.