Mental health in the two years following hypertensive and normotensive pregnancy: The Postpartum, Physiology, Psychology and Paediatric follow-up (P4) cohort study.

BACKGROUND: Postpartum mental illnesses and hypertensive disorders of pregnancy (HDP) are both common, and both associated with adverse maternal and child health outcomes. However, the relationship between them is unclear. This study aimed to investigate prevalence and symptom severity of depression, anxiety, and post-traumatic stress disorder (PTSD) 2-years postpartum in women with normal blood pressure (NBP) during pregnancy versus preeclampsia or gestational hypertension (GH). METHODS: Two-years follow-up of the prospective Postpartum, Physiology, Psychology and Paediatric (P4) Cohort Study was conducted in metropolitan Australia. Prevalence and symptom severity of depression (Edinburgh Postnatal Depression Scale, EPDS > 12), anxiety (7-item Generalized Anxiety Disorder scale, GAD-7 ≥ 10) and PTSD (Posttraumatic stress Diagnostic Scale, PDS/PDS-5) were measured and calculated for women with NBP, preeclampsia and GH. RESULTS: Among 365 participants (NBP: n = 271, preeclampsia: n = 75, GH: n = 19), 2-years postpartum depression prevalence was 3.9% (95% CI 2.3-6.4%): 4.4% after NBP, and 2.7% after preeclampsia (p = 0.53). Anxiety prevalence was higher after GH than NBP (15.8% versus 3.3%, p = 0.02). Prevalence of any mental illness (depression/anxiety/PTSD) was 5.9% (95% CI 3.8-8.8%); 5.6% after NBP, 4.1% after PE, and 15.8% after GH (p = 0.15). Although PTSD prevalence was low (1.4%), and similar between groups (p = 0.97), around 3 times more women after PE (8.1%), compared to NBP (2.5%), recalled childbirth as traumatic (p = 0.003). CONCLUSIONS: Preeclampsia, although associated with persistent perceptions of traumatic childbirth, did not alter the risk of mental illnesses at 2-years postpartum. GH (albeit in a small subgroup) was associated with increased anxiety scores. Larger, multicentre studies are required to clarify relationships between HDP and postpartum mental illness. TRIAL REGISTRATION: Retrospectively registered on 18/11/2013 with the Australian and New Zealand Clinical Trials Registry. REGISTRATION NUMBER: ACTRN 12613 00,126 0718.

Preeclampsia-Associated Cardiovascular Risk Factors 6 Months and 2 Years After Pregnancy: The P4 Study.

BACKGROUND: Increased cardiovascular risk following preeclampsia is well established and there are signs of early cardiovascular aging 6 months postpartum. This study assessed whether blood pressure (BP) and other cardiovascular measures are abnormal 2 years postpartum in the same cohort to determine ongoing risk markers. METHODS: Six months and 2 years postpartum, BP was measured using sphygmomanometry, 24-hour ambulatory BP monitoring, and noninvasive central BP. Anthropometric measures, blood, and urine biochemistry were performed. Cross-sectional comparisons between preeclampsia and normotensive pregnancy (NP) groups and longitudinal comparisons within each group were made at 6 months and 2 years. RESULTS: Two years postpartum, 129 NP, and 52 preeclampsia women were studied who also had 6 months measures. At both time points, preeclampsia group had significantly higher BP (office BP 2 years, 112±12/72±8 versus 104±9/67±7 mm Hg NP; [P<0.001]; mean ambulatory BP monitoring 116±9/73±8 versus 106±8/67±6 mm Hg NP; [P<0.001]). No significant BP changes noted 6 months to 2 years within either group. Office BP thresholds of 140 mm Hg systolic and 90 mm Hg diastolic classified 2% preeclampsia and 0% NP at 2 years. American Heart Association 2017 criteria (above normal, >120/80 mm Hg) classified 25% versus 8% (P<0.002), as did our reference range threshold of 122/79 mm Hg. American Heart Association criteria classified 60% post-preeclampsia versus 16% after NP with above-normal ambulatory BP monitoring (P<0.001). Other cardiovascular risk markers more common 2 years post-preeclampsia included higher body mass index (median 26.6 versus 23.1, P=0.003) and insulin resistance. CONCLUSIONS: After preeclampsia, women have significantly higher BP 6 months and 2 years postpartum, and have higher body mass index and insulin-resistance scores, increasing their future cardiovascular risk. Regular cardiovascular risk screening should be implemented for all who have experienced preeclampsia.

Preferences of Australian healthcare providers regarding education on long-term health after hypertensive disorders of pregnancy: a qualitative study.

OBJECTIVES: To explore Australian healthcare providers' (HCPs') preferred content, format and access to education regarding long-term health after hypertensive disorder of pregnancy (HDP), in order to guide the development of education programmes. DESIGN AND SETTING: A qualitative study using a framework analysis was undertaken. Registered HCP who were practising in Australia and previously completed a survey about long-term health after HDP were invited to participate. PARTICIPANTS: Twenty HCP were interviewed, including midwives, specialist obstetrician/gynaecologists, general practitioners with a diploma in obstetrics and gynaecology, and cardiologists. PRIMARY AND SECONDARY OUTCOME MEASURES: Exploration of preferred content, format and distribution of educational material post-HDP. RESULTS: Twenty HCP were interviewed in April to May 2020. Four main categories were identified. 'Obtaining evidence-based information for own learning' addressing own learning with preference for multi-disciplinary education, preferably endorsed or facilitated by professional organisations. 'Optimising the referral process from hospital to community health services' was about the need for structured long-term follow-up to transition from hospital to community health and align with HDP guidelines. 'Facilitating women's health literacy' addressed the need for evidence-based, print or web-based material to assist risk discussions with women. 'Seizing educational opportunities' addressed the responsibility of all HCP to identify education opportunities to initiate key health discussions with women. CONCLUSIONS: HCP provided ideas on content, format and access of education regarding long-term health post-HDP within the parameters of the Australian healthcare context. This evidence will guide educational developments for HCP on post-HDP health to ensure they can better care for women and families.

Maternal Adiposity and Energy Balance After Normotensive and Preeclamptic Pregnancies.

BACKGROUND: Preeclampsia is a major pregnancy complication associated with long-term maternal cardiometabolic disease. Research generally is focused on metabolic and pathophysiological changes during pregnancy; however, there is much less focus on the early postpartum period in subjects who suffered preeclampsia. The aim of this study was to (1) characterize energy intake and expenditure 6 months following normotensive and preeclamptic pregnancies and (2) examine associations between energy balance, body composition, insulin resistance measures (HOMA-IR), and clinical characteristics. DESIGN: A cross-sectional study 6 months following normotensive (n = 75) and preeclamptic (n = 22) pregnancies was performed. Metabolic measurements included anthropometrics measures, body composition via bioelectrical impedance analysis, 24-h energy expenditure via SenseWear Armbands, energy intake via a 3-day food diary, and serum metabolic parameters. RESULTS: Six months following preeclampsia, women had a significantly higher weight (77.3 ± 20.9 kg vs 64.5 ± 11.4 kg, P = 0.01), fat mass percentage (FM%; 40.7 ± 7.4% vs 34.9 ± 8.1%, P = 0.004), and insulin resistance (HOMA-IR 2.2 ± 1.5 vs 1.0 ± 0.7, P = 0.003), as well as reduced HDL levels (1.5 ± 0.4 mmol/L vs 1.8 ± 0.4 mmol/L, P = 0.01) compared to normotensive women. Women post-preeclampsia had lower activity-related energy expenditure (P = 0.02) but a decreased total energy intake (P = 0.02), leading to a more negative energy balance compared to their normotensive counterparts (-1942 kJ/24 h vs -480 kJ/24 h, P = 0.02). CONCLUSION: Increases in insulin resistance and FM%, reduced high-density lipoprotein, and more sedentary lifestyles characterize the postpartum period following preeclamptic compared with normotensive pregnancies. Early post-preeclampsia interventions, such as lifestyle behavior change, should be implemented and assessed to determine whether they reduce long-term cardiometabolic risk in women who experienced preeclampsia during pregnancy.

Assessing Australian women's knowledge and knowledge preferences about long-term health after hypertensive disorders of pregnancy: a survey study.

OBJECTIVES: To (1) assess women's current knowledge regarding long-term cardiovascular health after hypertensive disorders of pregnancy (2) elicit women's preferred educational content and format regarding health after hypertensive disorders of pregnancy. DESIGN AND SETTING: A custom-created online survey exploring Australian women's knowledge about long-term health after hypertensive disorders of pregnancy, distributed through consumer groups and social media. PARTICIPANTS: 266 women with (n=174) or without (n=92) a history of hypertensive disorders of pregnancy. PRIMARY AND SECONDARY OUTCOME MEASURES: (1) Proportion of women identifying long-term health risks after hypertensive disorder of pregnancy using a 10-point risk knowledge score with 0-4 'low', 4.1-7.0 'moderate' and 7.1-10 'high'. (2) Exploration of preferred content, format and distribution of educational material post hypertensive disorder of pregnancy. RESULTS: Knowledge scores about health after hypertensive disorder of pregnancy were moderate in groups with and without a history of the disorder. Knowledge was highest regarding risk of recurrent hypertensive disorders in a subsequent pregnancy, 'moderate' for chronic hypertension and heart attack, 'moderate' and 'low' regarding risk of heart disease and 'low' for diabetes and renal disease. Only 36% of all participants were aware that risks start within 10 years after the affected pregnancy. The majority of respondents with a history of hypertensive disorder of pregnancy (76%) preferred receiving information about long-term health 0-6 months post partum from a healthcare provider (80%), key organisations (60%), social media (47%) and brochures/flyers (43%). CONCLUSIONS: Women's knowledge regarding health risks after hypertensive disorder of pregnancy was 'moderate', although with important disease-specific gaps such as increased risk of diabetes. Most women wanted to be informed about their long-term health from a healthcare provider.

Microbiome Understanding in Maternity Study (MUMS), an Australian prospective longitudinal cohort study of maternal and infant microbiota: study protocol.

INTRODUCTION: Pregnancy induces significant physiological and cardiometabolic changes, and is associated with alterations in the maternal microbiota. Increasing rates of prepregnancy obesity, metabolic abnormalities and reduced physical activity, all impact negatively on the microbiota causing an imbalance between the commensal microorganisms (termed dysbiosis), which may drive complications, such as gestational diabetes or hypertensive disorders. Considerable work is needed to define the inter-relationships between the microbiome, nutrition, physical activity and pregnancy outcomes. The role of the microbiota during pregnancy remains unclear. The aim of the study is to define microbiota signatures longitudinally throughout pregnancy and the first year post birth, and to identify key clinical and environmental variables that shape the female microbiota profile during and following pregnancy. METHODS AND ANALYSIS: The Microbiome Understanding in Maternity Study (MUMS) is an Australian prospective longitudinal cohort study involving 100 mother-infant pairs. Women are enrolled in their first trimester and followed longitudinally. Assessment occurs at <13+0, 20+0-24+6 and 32+0-36+6 weeks gestation, birth and 6 weeks, 6 months and 12 months postpartum. At each assessment, self-collected oral, vaginal and faecal samples are collected with an additional postpartum skin swab and breastmilk sample. Each infant will have oral, faecal and skin swab samples collected. Measurements include anthropometrics, body composition, blood pressure, serum hormonal and metabolic parameters and vaginal pH. Dietary intake, physical activity and psychological state will be assessed using validated self-report questionnaires, and pregnancy and infant outcomes recorded. Parametric and non-parametric hypothesis tests will be used to test the association between high-risk and low-risk pregnancies and their outcomes. ETHICS AND DISSEMINATION: The study received the following approval: South Eastern Sydney Local Health District Research Ethics Committee (17/293 (HREC/17/POWH/605). Results will be made available to the participants of MUMS, their families and the funding bodies; in the form of a summary document. Results for the greater maternity care community and other researchers will be disseminated through conferences, local, national and international presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: ACTRN12618000471280 (prospectively registered).

Pregnancy with gestational hypertension or preeclampsia: A qualitative exploration of women's experiences.

BACKGROUND: Hypertension complicates 10% of pregnancies and involves specialised care of the woman and her baby, a longer stay in hospital, and an increased risk of physical and mental morbidity. There is limited research reporting the woman's perspective on her experience, how she coped with it psychologically, and whether the care she received influenced her experience. AIM: To gain insight into women's experience of hypertension in pregnancy and to report on what mediating factors may help improve their experience. METHODS: A qualitative descriptive study was undertaken. Data were collected through a semi-structured, face to face interview at 10-12 months postpartum. In total, 20 women who had experienced hypertension in their pregnancy were interviewed. Thematic analysis was used to analyse the data. FINDINGS: Four main themes were identified. These were: Reacting to the diagnosis, Challenges of being a mother, Processing and accepting the situation, and Moving on from the experience. The mediating factors that improved the experience were Feeling safe and trusting the care providers, Having continuity of care and carer, and Valuing social support from partner, family and friends. CONCLUSION: The diagnosis of hypertension in pregnancy has a significant impact on women. This affects their pregnancy and birth experience and their pathway to motherhood. The implications of the findings for midwifery practice include having access to multidisciplinary continuity models of care and facilitating the support for these women.

Comparisons of auscultatory hybrid and automated sphygmomanometers with mercury sphygmomanometry in hypertensive and normotensive pregnant women: parallel validation studies.

OBJECTIVE: To find a suitable replacement for mercury sphygmomanometry to measure blood pressure (BP) accurately in normal and hypertensive pregnancy. METHODS: Two parallel validation studies were carried out in 340 pregnant women, 170 with a hypertensive disorder and 170 normotensive women. An auscultatory hybrid sphygmomanometer, A&D UM-101, and a professional automated oscillometric device for office and clinic use, Omron HEM-907, were tested. Using a modified British Hypertension Society (BHS) Protocol, nine sequential BP recordings were taken alternating between the mercury sphygmomanometer and the study device. The first readings for each device were discarded, and three differences between mercury and study device were calculated for each woman for SBP and DBP. Main outcome measures were the percentages of BP readings that were within 5, 10 and 15 mmHg absolute difference from mercury sphygmomanometry. RESULTS: Women in both studies were an average of 34 weeks' gestation and of similar ethnicity, age and BMI. In hypertensive women, 29% had preeclampsia and 73% were receiving antihypertensives. Amongst hypertensive women, SBP was within 5 mmHg of mercury BP in 94% of readings with the auscultatory device and 75% with the automated device (P = 0.021); DBP was within 5 mmHg in 97 and 61% readings, respectively (P = 0.001). Results were similar amongst normotensive pregnant women. Both devices achieved an A/A rating according to the BHS protocol. CONCLUSION: The auscultatory hybrid sphygmomanometer is more accurate than the automated oscillometric device in pregnancy, specifically in hypertensive pregnancies. It is an acceptable replacement for mercury sphygmomanometry in pregnancy.

The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain.

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

An Arabidopsis reticulon and the atlastin homologue RHD3-like2 act together in shaping the tubular endoplasmic reticulum.

The endoplasmic reticulum (ER) is a network of membrane sheets and tubules connected via three-way junctions. A family of proteins, the reticulons, are responsible for shaping the tubular ER. Reticulons interact with other tubule-forming proteins (Dp1 and Yop1p) and the GTPase atlastin. The Arabidopsis homologue of Dp1/Yop1p is HVA22. We show here that a seed-specific isoform of HVA22 labels the ER in tobacco (Nicotiana tabacum) cells but its overexpression does not alter ER morphology. The closest plant homologue of atlastin is RHD3. We show that RHD3-like 2 (RL2), the seed-specific isoform of RHD3, locates to the ER without affecting its shape or Golgi mobility. Expression of RL2-bearing mutations within its GTPase domain induces the formation of large ER strands, suggesting that a functional GTPase domain is important for the formation of three-way junctions. Coexpression of the reticulon RTNLB13 with RL2 resulted in a dramatic alteration of the ER network. This alteration did not depend on an active GTPase domain but required a functional reticulon, as no effect on ER morphology was seen when RL2 was coexpressed with a nonfunctional RTNLB13. RL2 and its GTPase mutants coimmunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 act together in modulating ER morphology.

Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p.

BACKGROUND: Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae. METHODOLOGY AND PRINCIPAL FINDINGS: Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors. CONCLUSIONS AND SIGNIFICANCE: The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.

A prospective randomized study of automated versus mercury blood pressure recordings in hypertensive pregnancy (PRAM Study).

BACKGROUND: Mercury sphygmomanometry is being replaced with automated blood pressure (BP) recording. We tested the potential impact of this change on the outcomes of pregnant women with hypertension. METHODS: Following routine detection of hypertension by mercury sphygmomanometry, 220 pregnant women with hypertension were randomized to have all subsequent BPs recorded with either mercury sphygmomanometry or an automated BP device (Omron HEM-705CP) for the remainder of their pregnancy. We used a prospective randomized open study with blinded end points design. MAIN OUTCOME MEASURES: The primary maternal outcome measure was the number of women having any episode of severe hypertension (BP 170/110 mmHg). The primary fetal outcome measure was small for gestational age baby. RESULTS: Both groups were similar in age, weight, parity, race, use of assisted reproductive technology before this pregnancy, diabetes prevalence, smoking, mercury BP in the first trimester, use of antihypertensives at the time of study entry, and type of hypertension (essential, gestational or preeclampsia) at study entry. Severe hypertension occurred in 39% of women using mercury sphygmomanometry and 44% using the automated device (p = 0.5). Small for gestational age rates were 12 and 17%, respectively (p = 0.3). About 47% of women had preeclampsia at delivery. Birth weight and perinatal mortality were similar between groups. CONCLUSIONS: Using this automated BP recording device in hypertensive pregnant women is associated with similar maternal and fetal outcomes as in women whose BP is measured by sphygmomanometry, despite intra-individual BP differences between the two methods of recording BP.

An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis.

We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.

RNAi screening of Drosophila (Sophophora) melanogaster S2 cells for ricin sensitivity and resistance.

The ribosome-inhibiting toxin ricin binds exposed ß1→4 linked galactosyls on multiple glycolipids and glycoproteins on the cell surface of most eukaryotic cells. After endocytosis, internal cell trafficking is promiscuous, with only a small proportion of ricin proceeding down a productive (cytotoxic) trafficking route to the endoplasmic reticulum (ER). Here, the catalytic ricin A chain traverses the membrane to inactivate the cytosolic ribosomes, which can be monitored by measuring reduction in protein biosynthetic capacity or cell viability. Although some markers have been discovered for the productive pathway, many molecular details are lacking. To identify a more comprehensive set of requirements for ricin intoxication, the authors have developed an RNAi screen in Drosophila S2 cells, screening in parallel the effects of individual RNAi treatments alone and when combined with a ricin challenge. Initial screening of 806 gene knockdowns has revealed a number of candidates for both productive and nonproductive ricin trafficking, including proteins required for transport to the Golgi, plus potential toxin interactors within the ER and cytosol.

LG186: An inhibitor of GBF1 function that causes Golgi disassembly in human and canine cells.

Brefeldin A-mediated inhibition of ADP ribosylation factor (Arf) GTPases and their guanine nucleotide exchange factors, Arf-GEFs, has been a cornerstone of membrane trafficking research for many years. Brefeldin A (BFA) is relatively non-selective inhibiting at least three targets in human cells, Golgi brefeldin A resistance factor 1 (GBF1), brefeldin A inhibited guanine nucleotide exchange factor 1 (BIG1) and brefeldin A inhibited guanine nucleotide exchange factor 2 (BIG2). Here, we show that the previously described compound Exo2 acts through inhibition of Arf-GEF function, but causes other phenotypic changes that are not GBF1 related. We describe the engineering of Exo2 to produce LG186, a more selective, reversible inhibitor of Arf-GEF function. Using multiple-cell-based assays and GBF1 mutants, our data are most consistent with LG186 acting by selective inhibition of GBF1. Unlike other Arf-GEF and reported GBF1 inhibitors including BFA, Exo2 and Golgicide A, LG186 induces disassembly of the Golgi stack in both human and canine cells.

Fine tuning Exo2, a small molecule inhibitor of secretion and retrograde trafficking pathways in mammalian cells.

The small molecule 4-hydroxy-3-methoxybenzaldehyde (5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone (Exo2) stimulates morphological changes at the mammalian Golgi and trans-Golgi network that are virtually indistinguishable from those induced by brefeldin A. Both brefeldin A and Exo2 protect cells from intoxication by Shiga(-like) toxins by acting on other targets that operate at the early endosome, but do so at the cost of high toxicity to target cells. The advantage of Exo2 is that it is much more amenable to chemical modification and here we report a range of Exo2 analogues produced by modifying the tetrahydrobenzothienopyrimidine core, the vanillin moiety and the hydrazone bond that links these two. These compounds were examined for the morphological changes they stimulated at the Golgi stack, the trans-Golgi network and the transferrin receptor-positive early endosomes and this activity correlated with their inherent toxicity towards the protein manufacturing ability of the cell and their protective effect against toxin challenge. We have developed derivatives that can separate organelle morphology, target specificity, innate toxicity and toxin protection. Our results provide unique compounds with low toxicity and enhanced specificity to unpick the complexity of membrane trafficking networks.

Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum.

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA(Delta)), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER-Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA(Delta), although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.

Disulfide formation in plant storage vacuoles permits assembly of a multimeric lectin.

The ER (endoplasmic reticulum) has long been considered the plant cell compartment within which protein disulfide bond formation occurs. Members of the ER-located PDI (protein disulfide isomerase) family are responsible for oxidizing, reducing and isomerizing disulfide bonds, as well as functioning as chaperones to newly synthesized proteins. In the present study we demonstrate that an abundant 7S lectin of the castor oil seed protein storage vacuole, RCA (Ricinus communis agglutinin 1), is folded in the ER as disulfide bonded A-B dimers in both vegetative cells of tobacco leaf and in castor oil seed endosperm, but that these assemble into (A-B)2 disulfide-bonded tetramers only after Golgi-mediated delivery to the storage vacuoles in the producing endosperm tissue. These observations reveal an alternative and novel site conducive for disulfide bond formation in plant cells.

Simple oxidation of pyrimidinylhydrazones to triazolopyrimidines and their inhibition of Shiga toxin trafficking.

The oxidative cyclisation of a range of benzothieno[2,3-d]pyrimidine hydrazones (7a-j) to the 1,2,4-triazolo[4,3-c]pyrimidines (8a-j) catalysed by lithium iodide or to the 1,2,4-triazolo[1,5-c]pyrimidines (10a-j) with sodium carbonate is presented. A complementary synthesis of the 1,2,4-triazolo[1,5-c]pyrimidines starting from the amino imine 11 is also reported. The effect of these compounds on Shiga toxin (STx) trafficking in HeLa cells and comparison to the previously reported Exo2 is also detailed.

Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.