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.

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.

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.

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.

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.

Placental P-glycoprotein is unaffected by timing of antenatal glucocorticoid therapy but reduced in SGA preterm infants.

INTRODUCTION: The beneficial effects of antenatal glucocorticoid therapy on fetal lung maturation require their passage across the placental glucocorticoid barrier, composed of glucocorticoid metabolising enzymes, such as 11 beta hydroxysteroid dehydrogenase (11ßHSD), and proteins that efflux glucocorticoids, such as P-glycoprotein (P-gp). We have shown that 11ßHSD2 activity is responsive to antenatal glucocorticoids, however the effect on placental P-gp remains unknown. Since antenatal glucocorticoids have a greater prophylactic effect in females compared to males, we also assessed whether this therapy induced sexually dimorphic effects on P-gp expression, as well as on placental inflammatory processes mediated by corticosteroids. METHODS: Placentas were collected from 53 women presenting in threatened preterm labour, and processed to assess cytokine and P-gp mRNA expression, as well as P-gp localisation using immunohistochemistry. RESULTS: Placental cytokine, P-gp mRNA and protein expression were not altered by timing of antenatal glucocorticoids or fetal sex. However, both P-gp mRNA and protein expression were significantly reduced in placentas from infants born small for gestational age (SGA) compared to appropriately grown infants (p < 0.05), suggesting a role for P-gp in its pathogenesis via the provision of a net increase in fetal exposure to bioactive exogenous glucocorticoids. CONCLUSIONS: While this study identified no change in placental P-gp following antenatal glucocorticoids, it has provided evidence that P-gp plays an important role in cases of SGA. This supports the known mechanistic relationship between antenatal glucocorticoids, fetal development and the postnatal phenotype. Given that P-gp also confers fetal protection from a number of drugs, this finding warrants further investigation to improve clinical management of the SGA fetus.

Localisation and characterisation of uncoupling protein-2 (UCP2) in the human preterm placenta.

The increase in oxidative stress during pregnancy is associated with increased placental antioxidant enzyme activity and may additionally be limited by the uncoupling proteins (UCPs). There is little data on the expression and localisation of UCP2 in the human preterm placenta or on its role in the regulation of placental oxidative stress. Placentae were collected from women with singleton pregnancies who delivered between 24 and 36 weeks gestation (n = 54) and from a term reference group who delivered following uncomplicated pregnancy (n = 11). UCP2 expression and localisation was determined by quantitative real-time RTPCR using Taqman gene expression assays and immunohistochemistry. Placental lipid hydroperoxide and nitrotyrosine content was determined by ELISA. UCP2 mRNA expression increased from 24 to 41 weeks gestation (p < 0.001) and was positively correlated with placental weight (p = 0.004). While UCP2 expression was lower in small for gestational age infants (p = 0.045) it did not differ with respect to timing of antenatal betamethasone exposure nor with placental lipid hydroperoxide or nitrotyrosine content. UCP2 staining was identified in the cytotrophoblast in 34% of samples and in the syncytiotrophoblast in 63% of samples. Cytotrophoblast staining was more frequent in later gestations (p = 0.03) with syncytiotrophoblast UCP2 staining was not altered by gestation. In the preterm group, no association was observed with time since antenatal betamethasone exposure or placental lipid hydroperoxide or nitrotyrosine content. The current data supports gestation dependant alterations in UCP2 mRNA expression and immunohistochemical localisation in the human placenta but no evidence for an important role for UCP2 in protection against placental oxidative damage.

Review: The feto-placental unit, pregnancy pathology and impact on long term maternal health.

Pregnancy induces a number of alterations to maternal physiology to accommodate the increased demands made by the developing fetus and placenta. These alterations appear at least in part to be driven by products derived from the feto-placental unit, including microchimeric cells, as well as placental exosomes and microparticles, inducing changes to maternal physiology both during pregnancy and beyond. Further, increasing evidence suggests that some of these alterations are dependent on the sex of the fetus. Pre-eclampsia and asthma represent two common pregnancy complications that have provided valuable insight into how the feto-placental unit influences maternal physiology in a sex-specific manner. Pregnancy-induced alterations in maternal physiology may expose pre-existing subclinical pathologies and provide insight into future maternal health and disease. While most pregnancy-induced alterations to the maternal system are reversed following delivery, some can persist after parturition leading to cardiovascular, metabolic and autoimmune disease and increased risk of early mortality.

Effect of co-infusion of dextrose-containing solutions on red blood cell haemolysis during packed red cell transfusion.

AIM: Transfusion guidelines prohibit co-infusion of maintenance intravenous fluid solutions, with significant consequences for neonatal clinical care. This study investigated co-infusion-related haemolysis in an in vitro model closely resembling clinical practice. METHODS: Packed red blood cells (PRBCs, n=8) were co-infused at 5 and 10 ml/h with dextrose 5%, 10% and intravenous amino acid solution (synthamin). Free haemoglobin (fHb), as a measure of haemolysis, was measured by spectrophotometry and presented as % haemolysis and total fHb content (µmol/l). RESULTS: Following co-infusion, there was no significant increase in PRBC haemolysis with either type of solution co-infused (p=0.82) or infusion rate (p=0.5). Neither macroscopic nor microscopic agglutination was observed during co-infusion for any type of solution co-infused. CONCLUSIONS: Co-infusion does not result in increased haemolysis, with total fHb significantly lower than currently accepted safe thresholds for fHb. Adherence to current guidelines may place undue restrictions on current transfusion practice in neonatal intensive care.

Haemoglobin transfusion threshold in very preterm newborns: a theoretical framework derived from prevailing oxygen physiology.

The overwhelming majority of very preterm newborns receive at least one transfusion during their in hospital stay. However, despite two recent randomised trials, the appropriate haemoglobin transfusion threshold in these high risk infants remains unclear. Typically, clinicians consider gestation, chronologic age and illness severity in order to determine the need for transfusion. There is, however, no simple way to balance these heterogeneous variables in order to arrive at a transfusion threshold without considering the prevailing oxygen physiology. This is particularly important during the transition to extra-uterine life, a time when the risk of brain injury is highest. We hypothesise that dysregulated cerebral oxygen handling, characterised by restricted oxygen consumption from suboptimal oxygen delivery increases the risk of hypoxic ischaemic brain injury in very preterm newborns and is the single common patho-physiologic process underlying early acquired brain injury in the preterm newborn. Our proposed framework, based on the physiology of oxygen handling, considers the prevailing oxygen kinetics in the very preterm newborn as a means of deriving the appropriate Hb transfusion threshold thereby balancing oxygen delivery and consumption and avoiding hypoxic ischaemic early brain injury. Manipulation of the oxygen delivery equation, with consideration of the likely chronologic changes to cardiac output in particular, permit derivation of a transfusion threshold in the first week of life and represents a novel therapeutic intervention aimed solely at prevention of early acquired brain injury and its associated long term neuro-developmental burden.

Influence of sex and glucocorticoid exposure on preterm placental pro-oxidant-antioxidant balance.

Glucocorticoids (GC) are known to influence fetal ROS production and anti-oxidant defences yet little attention has focused on the potential for effects in the placenta. We hypothesised that antenatal GC exposure alters placental pro-oxidant-anti-oxidant balance sex-specifically, based upon the known relationship between male sex and poor pregnancy outcome. Placentae were collected from 60 women who delivered between 24 and 31 completed weeks gestation and placental oxidative and nitrative stress (protein carbonyl, lipid hydroperoxide, and nitrotyrosine concentration) and anti-oxidant enzyme activity (glutathione peroxidase, thioredoxin reductase, and superoxide dismutase) measured. A pro-oxidant state was observed in placentae of male compared to female infants born within 72 h of antenatal GC exposure, with higher levels of protein carbonyl content (p = 0.04), lipid hydroperoxide (p < 0.01) and nitrotyrosine content (p = 0.02), and lower levels of glutathione peroxidase activity (p = 0.01). A pro-oxidant state continued to be observed in placentae of males compared to females born outside of 72 h, with higher protein carbonyl content (p = 0.04) and lower glutathione peroxidase activity (p = 0.01) than females, however no differences in placental lipid hydroperoxide and nitrotyrosine content were observed. These sex-specific alterations in products of placental oxidative stress could not purely be explained by differences in clinical illness severity (CRIB2 score). Therefore, these sex-specific alterations in placental pro-oxidant-antioxidant balance in response to antenatal betamethasone exposure, independent of illness severity, could contribute to the patho-physiologic processes underlying oxygen radical diseases of the newborn, conditions known to exhibit a male excess.

The presence of maternal asthma during pregnancy suppresses the placental pro-inflammatory response to an immune challenge in vitro.

The mechanisms that contribute to adverse outcomes for the neonate in pregnancies complicated by asthma may be mediated via changes in placental immune function. This study was designed to determine whether the presence of maternal asthma during pregnancy alters the placental pro-inflammatory immune response in vitro. A prospective cohort study of women with asthma (n = 22) and control (n = 11) subjects had placentae collected immediately after delivery. Placental explants were exposed to an immune challenge, lipopolysaccharide, in the presence and absence of cortisol in vitro. Cytokines, glucocorticoid receptor α (GR α) and p38 MAPK protein were measured. Placentae of control pregnancies had an increase in pro-inflammatory cytokine production over a 24 h period. Placentae from pregnancies complicated by maternal asthma had a reduced pro-inflammatory cytokine response to an immune challenge relative to the controls especially in relation to the production of interleukin (IL)-1ß and TNFα regardless of fetal sex. Cortisol inhibition of placental cytokine production was dependent on timing of exposure, fetal sex and presence and absence of asthma. GRα and p38 MAPK protein expression did not appear to contribute to differences in response to endotoxin or cortisol. Maternal asthma during pregnancy induces a hyposensitive inflammatory state in the placenta which is regulated by cortisol in a sexually dimorphic manner.

Microvascular flow, clinical illness severity and cardiovascular function in the preterm infant.

OBJECTIVES: To characterise the relationships between peripheral microvascular blood flow and measures of physiological and cardiovascular function in preterm infants in the immediate newborn period. DESIGN: Prospective observational cohort study. SETTING: Tertiary neonatal intensive care unit, New South Wales, Australia. PATIENTS: Ninety-six preterm neonates (24-36 weeks' gestation) admitted to the neonatal intensive care unit. MAIN OUTCOME MEASURE: Relationship between laser Doppler-derived basal microvascular blood flow, functional echocardiographic measurements of cardiovascular status, mean arterial blood pressure and clinical illness severity at 24, 72 and 120 h of age. RESULTS: At 24 h of age, multiple linear regression revealed a significant positive relationship, independent of gestational age, between baseline microvascular blood flow and clinical risk index for babies (CRIB II) score (r2 = 0.442). Microvascular blood flow was inversely related to mean arterial blood pressure (r2 = -0.563), and correlated positively with left ventricular output (r2 = 0.435). Microvascular blood flow continued to exhibit a significant inverse relationship with mean arterial blood pressure (r2 = -0.4) at 72 h of age, but by 120 h no significant relationships were evident. CONCLUSIONS: This is the first study to show that baseline microvascular blood flow in premature infants exhibits significant relationships with clinical illness severity and cardiovascular function in the immediate postnatal period. The effects of temporal and functional changes in the microvasculature on cardiovascular adaptation warrant further detailed study.

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.

Screening and characterization of microbial inhibitors against eukaryotic protein phosphatases (PP1 and PP2A).

AIM: To identify novel microbial inhibitors of protein phosphatase 1 (PP1). METHODS AND RESULTS: 750 actinomycetes and 408 microfungi were isolated from Sabah forest soils and screened for production of potential PP1 inhibitors using an in vivo screening system, in which candidate inhibitors were identified through mimicking the properties of PP1-deficient yeast cells. Acetone extracts of two fungi, H9318 (Penicillium) and H9978 (non-Penicillium) identified in this way showed inhibitory activity towards both mammalian PP1 and PP2A in an in vitro phosphatase assay, while extract from H7520 (Streptomyces) inhibited PP2A but not PP1. Consistently, using a drug-induced haploinsufficiency test, strains with either reduced PP1 or PP2A function were hypersensitive to H9318 and H9978 extracts whereas only the latter strain showed hypersensitivity to H7250 extract. H9318 extract was fractionated using RP-HPLC into two active peaks (S1 and S2). A yeast strain with reduced PP1 function showed hypersensitivity to fraction S2 whereas a strain with reduced PP2A function was hypersensitive to fraction S1. However, S1 and S2 inhibited both PP1 and PP2A activities to a similar extent. CONCLUSION: Three candidate PP inhibitors have been identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Further development may generate useful research tools and ultimately therapeutic agents.

A prospective study of household smoking bans and subsequent cessation related behaviour: the role of stage of change.

OBJECTIVE: To assess the degree to which smokers living with a full household ban on smoking change their cessation related behaviour. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study; follow up of a population based cohort of 1133 smokers, identified from a 1997 telephone survey of adult Oregonians. After a median of 21 months, 565 were located and reinterviewed. MAIN OUTCOME MEASURES: Quit attempts, time until relapse, and smoking cessation, defined as seven day and 90 day sustained abstinence at follow up. RESULTS: A full ban at baseline was associated with a doubling of the odds of a subsequent quit attempt (odds ratio (OR) = 2.0, 95% confidence interval (CI), 1.0 to 3.9). Among respondents in the preparation stage at baseline (intention to quit in the next month with a quit attempt in the previous year), a full ban was associated with a lower relapse rate (hazard ratio = 0.5 (95% CI, 0.2 to 0.9)), while for those in precontemplation/contemplation (no intention to quit or intention to quit within the next six months, respectively), there was no significant association between full ban and relapse rate. For respondents in preparation, those with a full ban had over four times the odds of being in cessation for seven or more days before the follow up call (OR = 4.4 (1.1 to 18.7)), but for those in precontemplation/contemplation, full bans were unrelated to cessation. CONCLUSIONS: Full household bans may facilitate cessation among smokers who are preparing to quit by increasing quit attempts. They may also prolong time to relapse among those smokers.

Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.

The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild-type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator-dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy-terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor.

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle. Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest. The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells.

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations.

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.