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Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Boston Scientific ISR lite. Background In carefully selected patients, Implantable Cardioverter Defibrillators reduce all-cause mortality by reducing the burden of sudden cardiac death. Most patients who have an ICD implant do not receive life-prolonging therapy from it. Little research has been undertaken to determine which patients benefit the least from ICD therapy. As patients age and accumulate comorbidities, the risk of death increases and the benefit of ICDs diminishes. Purpose We sought to evaluate the impact of comorbidity, frailty, functional status on death prior to ICD therapy as this group has the least to gain from ICD therapy. Methods A prospective, multicentre, observational study involving 15 English hospitals was undertaken. Patients were eligible for inclusion for the study if they were scheduled to have a de novo, replacement or an upgrade to a high-energy device (transvenous or subcutaneous ICD or CRT-D). Baseline characteristics were collected including age, gender, device type, LV function, medication, baseline bloods if available and were asked to complete a frailty assessment, Fried score; a functional status questionnaire (EQ5D5L), Charlson Co-morbidity Score and EQ5D5L data were calculated. Results In total, 662 patients were enrolled, mean age 65.6 (IQR 65-75) years, 77.3% male, 55.9% de novo implants, 33.4% replacements and 10.5% upgrade. Overall, 32% were for a secondary prevention indication, 49.% had a single or dual chamber transvenous ICD, 45.3% had a CRT-D and 5% had an S-ICD) implanted. A total of 63 patients (9.5%) died across this period, of these, 54 died without receiving appropriate therapy. Frailty was present in 83/662 (12.5%) and severe co-morbidity was present in 69/662 (10.4%). The overall survival probability estimated to be 90.2% at 2.5 years, and was 68% in frail patients. Figure 1. Multivariate predictors of death with no appropriate therapy were identified and a risk score comprising frailty, severe co-morbidity, age >70 years, eGFR Conclusion Frailty and severe co-morbidity are powerful, independent predictors of death with no appropriate in ICD/CRT-D recipients.

Data sharing: The PHOSP-COVID study protocol, consent form, definition, and derivation of clinical characteristics and outcomes, training materials, regulatory documents, information about requests for data access, and other relevant study materials are available online. UK Biobank information can be released once necessary approvals have been obtained. Other data (eg, the R code and protocol) will be made available on reasonable request to the corresponding author. Copyright © 2023 The Author(s). Background: Sleep disturbance is common following hospital admission both for COVID-19 and other causes. The clinical associations of this for recovery after hospital admission are poorly understood despite sleep disturbance contributing to morbidity in other scenarios. We aimed to investigate the prevalence and nature of sleep disturbance after discharge following hospital admission for COVID-19 and to assess whether this was associated with dyspnoea. Methods: CircCOVID was a prospective multicentre cohort substudy designed to investigate the effects of circadian disruption and sleep disturbance on recovery after COVID-19 in a cohort of participants aged 18 years or older, admitted to hospital for COVID-19 in the UK, and discharged between March, 2020, and October, 2021. Participants were recruited from the Post-hospitalisation COVID-19 study (PHOSP-COVID). Follow-up data were collected at two timepoints: an early time point 2–7 months after hospital discharge and a later time point 10–14 months after hospital discharge. Sleep quality was assessed subjectively using the Pittsburgh Sleep Quality Index questionnaire and a numerical rating scale. Sleep quality was also assessed with an accelerometer worn on the wrist (actigraphy) for 14 days. Participants were also clinically phenotyped, including assessment of symptoms (ie, anxiety [Generalised Anxiety Disorder 7-item scale questionnaire], muscle function [SARC-F questionnaire], dyspnoea [Dyspnoea-12 questionnaire] and measurement of lung function), at the early timepoint after discharge. Actigraphy results were also compared to a matched UK Biobank cohort (non-hospitalised individuals and recently hospitalised individuals). Multivariable linear regression was used to define associations of sleep disturbance with the primary outcome of breathlessness and the other clinical symptoms. PHOSP-COVID is registered on the ISRCTN Registry (ISRCTN10980107). Findings: 2320 of 2468 participants in the PHOSP-COVID study attended an early timepoint research visit a median of 5 months (IQR 4–6) following discharge from 83 hospitals in the UK. Data for sleep quality were assessed by subjective measures (the Pittsburgh Sleep Quality Index questionnaire and the numerical rating scale) for 638 participants at the early time point. Sleep quality was also assessed using device-based measures (actigraphy) a median of 7 months (IQR 5–8 months) after discharge from hospital for 729 participants. After discharge from hospital, the majority (396 [62%] of 638) of participants who had been admitted to hospital for COVID-19 reported poor sleep quality in response to the Pittsburgh Sleep Quality Index questionnaire. A comparable proportion (338 [53%] of 638) of participants felt their sleep quality had deteriorated following discharge after COVID-19 admission, as assessed by the numerical rating scale. Device-based measurements were compared to an age-matched, sex-matched, BMI-matched, and time from discharge-matched UK Biobank cohort who had recently been admitted to hospital. Compared to the recently hospitalised matched UK Biobank cohort, participants in our study slept on average 65 min (95% CI 59 to 71) longer, had a lower sleep regularity index (–19%; 95% CI –20 to –16), and a lower sleep efficiency (3·83 percentage points; 95% CI 3·40 to 4·26). Similar results were obtained when comparisons were made with the non-hospitalised UK Biobank cohort. Overall sleep quality (unadjusted effect estimate 3·94; 95% CI 2·78 to 5·10), deterioration in sleep quality following hospital admission (3·00; 1·82 to 4·28), and sleep regularity (4·38; 2·10 to 6·65) were associated with higher dyspnoea scores. Poor sleep quality, deterioration in sleep quality, and sleep regularity were also associated with impaired lung function, as assessed by forced vital capacity. Depending on the sleep metric, anxiety mediated 18–39% of the effect of sleep disturbance on dyspnoea, while muscle weakness mediated 27–41% of this effect. Interpretation: Sleep disturbance following hospital admission for COVID-19 is associated with dyspnoea, anxiety, and muscle weakness. Due to the association with multiple symptoms, targeting sleep disturbance might be beneficial in treating the post-COVID-19 condition. Funding: UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council. UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council.

Patients with aortic aneurysms (AA) are often co-morbid and susceptible to frailty. Low core muscle mass has been used as a surrogate marker of sarcopenia and indicator of frailty. This study aimed to assess association between core muscle mass with sarcopenia screening tool SARC-F and Clinical Frailty Scale (CFS) in patients with AA.Prospective audit of patients in pre-operative aortic clinic between 01/07/2019-31/01/2020 including frailty assessment using Rockwood CFS and sarcopenia screening using SARC-F questionnaire. Psoas and sartorius muscle area were measured on pre-operative CT scans and adjusted for height. Association was assessed using Spearman's rank correlation coefficient.Of 84 patients assessed, median age was 75 years [72,82], 84.5% were men, 65.5% were multimorbid and 63.1% had polypharmacy. Nineteen percent were identified as frail (CFS score3) and 6.1% positively screened for sarcopenia (SARC-F score 4 or more). Median psoas area (PMA) at L3 was 5.6cm2/m2 [4.8,6.6] and L4 was 7.4cm2/m2 [6.3,8.6]. Median sartorius area (SMA) was 1.8 cm2/m2 [1.5,2.2]. CFS demonstrated weak but statistically significant negative correlation with height-adjusted PMA at L3 (r=-0.25, p=0.034) but not at L4 (r=-0.23, p=0.051) or with SMA (r=-0.22, p=0.065). No association was observed between SARC-F score and PMA or SMA (L3 PMA r=-0.015, p=0.9; L4 PMA r=-0.0014, p= 0.99; SMA r=-0.051, p=0.67).CFS showed higher association with CT-derived muscle mass than SARC-F. Comprehensive pre-operative risk-stratification tools which incorporate frailty assessment and body composition analysis may assist in decision making for surgery and allow opportunity for pre-habilitation.

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research.

Unlike annual crops where reclamation leaching of salts can be readily conducted between cropping, leaching of salts in permanent crops that are drip irrigated pose challenges. A need exists to formulate and test a management-type of salinity model for drip irrigation of table grapes. The model reported herein predicts the distribution of salts along the vine row and between the rows during the growing season, as affected by reactivity of salts of the applied irrigation water as well as rate and duration of drip application. The calibrated model reproduced the initial field salinity profiles after repeated irrigation cycles by adjusting only the routing factor α which is the ratio of horizontal to vertical water flow. After eight cycles the profiles stabilized and the calibrated horizontal to vertical flow routing ratio was 0.6. There is remarkable agreement between measured and simulated salinity. Corresponding soil moisture profiles show the expected high water content with depth at the emitter, the decrease in surface water content with radial distance and the increase with depth, at the distal end of the wedge. Although the model is location specific it can be applied knowing soil, initial and boundary conditions, as well as irrigation application quantity and quality and as such can be applied location by location in order to assess flow and quality of deep percolation recharging the groundwater system. With this capacity the model can predict soil water quantity and quality outcomes for possible land and water management scenarios.

In the armoury of the immune system developed to combat the various micro-organisms that could invade the host, the neutrophil forms the first line of defence against rapidly dividing bacteria and fungi. However, as humans age they become more susceptible to infection with these microbes and this has been ascribed to a decline in immune status, termed immune senescence. Here we summarize the literature specifically concerning the attenuation of neutrophil function with age and the possible mechanisms underlying their reduced response to infectious agents.

Several studies have attempted to identify regions of the MHC class II molecule that participate in signal transduction. The importance of intact murine I-A cytoplasmic domains, either to tether the class II molecule to the cytoskeleton or to recruit signal-transducing proteins, is now well established. Recent data have also suggested that residues of the I-A beta transmembrane are involved in a second distinct signaling pathway. In the light of data suggesting that the second messengers activated on ligation of human and mouse class II molecules may differ, we set out to investigate whether the structural requirements for signaling for the human DR molecule are similar to those already described for the murine I-A molecule. We show that mutant DR class II molecules lacking 12 amino acids of the beta-chain cytoplasmic tail fail to translocate the protein kinase C alpha (PKC alpha) and PKC beta II isoenzymes following stimulation with an anti-class II mAb. In contrast, truncation of either or both cytoplasmic domains of the DR molecule has no effect on class II-induced tyrosine kinase activity. PKC translocation following class II stimulation has been observed in both human and murine B lymphocytes, whereas tyrosine kinase activation is present in human B lymphocytes but absent in resting murine B lymphocytes. Therefore, we conclude that the DR beta cytoplasmic tail is requisite for the principal signaling pathway initiated via MHC class II. These data suggest that the signaling pathways seen in resting vs primed murine B cells may also operate in human APCs.

Ricin is an abundant protein component of Ricinus communis seeds (castor beans) that is exquisitely toxic to mammalian cells. It consists of an enzymic polypeptide that catalyzes the N-glycosidic cleavage of a specific adenine residue from 28S ribosomal RNA, joined by a single disulfide bond to a galactose (cell)-binding lectin. The enzymatic activity renders ribosomes containing depurinated 28S RNA incapable of protein synthesis. The bipartite molecular structure of ricin allows it to bind to the mammalian cell surface, enter via endocytic uptake, and deliver the catalytically active polypeptide into the cell cytosol where it irreversibly inhibits protein synthesis causing cell death. Because of its cytotoxic potency, modified ricin is being used for the selective killing of unwanted cells and for the toxigenic ablation of cell lineages in transgenic organisms.

Although pollinators are thought to select on flower colour, few studies have experimentally decoupled effects of colour from correlated traits on pollinator visitation and pollen transfer. We combined selection analysis and phenotypic manipulations to measure the effect of petal colour on visitation and pollen export at two spatial scales in Wahlenbergia albomarginata. This species is representative of many New Zealand alpine herbs that have secondarily evolved white or pale flowers. The major pollinators, solitary bees, exerted phenotypic selection on flower size but not colour, quantified by bee vision. When presented with manipulated flowers, bees visited flowers painted blue to resemble a congener over white flowers in large, but not small, experimental arrays. Pollen export was higher for blue flowers in large arrays. Pollinator preference does not explain the pale colouration of W. albomarginata, as commonly hypothesized. Absence of bright blue could be driven instead by indirect selection of correlated characters.

TheRicinus communis (castor bean) 2S albumin is a heterodimer of glutamine-rich, disulphidelinked 4 and 7 kDa polypeptides. A cDNA library was constructed using mRNA from maturing castor bean endosperm as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present in the 2 S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor included two further glutamine-rich sequences which, in terms of their size and conserved cysteine residues typically found in seed proteins of the 2S albumin superfamily, possibly represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme is proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. The generation of a second heterodimer seems likely since polypeptide candidates for its small and large subunits were found in theRicinus 2S albumin fraction, and N-terminal protein sequencing confirmed the existence of the putative small subunit.

The catalytic polypeptides of certain bacterial and plant protein toxins reach their substrates in the cytosol of mammalian cells by retro-translocation from the endoplasmic reticulum (ER). Emerging evidence indicates that these proteins subvert the ER-associated protein degradation (ERAD) pathway that normally removes misfolded or unassembled proteins from the ER, to achieve retrotranslocation. Upon entering the ER lumen, the toxins are unfolded to be perceived as ERAD substrates. Toxins that retro-translocate from the ER have an unusually low lysine content to avoid ubiquitin-mediated proteasomal degradation. This allows the exported toxins to refold into the proteasome-resistant, biologically active conformation, and leads to cellular intoxication.

Polycystic ovary syndrome (PCOS) is characterised by anovulation, hyperandrogenaemia and insulin resistance. Hyperinsulinaemia is known to be associated with an increase in cardiovascular risk and the development of diabetes mellitus. If insulin sensitising agents such as metformin are effective in treating features of PCOS, then they could have wider health benefits than just treating the symptoms of the syndrome.To assess the effectiveness of insulin sensitising drugs in improving clinical and biochemical features of PCOS.We searched the Cochrane Menstrual DisordersSubfertility Group trials register (December 2002), the Cochrane Central Register of Controlled Trials (Cochrane Library, Issue 4, 2002), MEDLINE (January 1966 to December 2002), and EMBASE (January 1985 to December 2002).Randomised controlled trials which investigated the effect of insulin sensitising drugs compared with either placebo or no treatment, or compared with an ovulation induction agent.Performed by two reviewers, one blinded to information that could have identified the authors, publisher or results of each study. Fifteen trials were included for analysis, 13 of them using metformin and involving 543 participants.Meta-analysis showed that metformin is effective in achieving ovulation in women with PCOS with odds ratios of 3.88 (CI 2.25 to 6.69) for metformin versus placebo and 4.41 (CI 2.37 to 8.22) for metformin and clomifene versus clomifene alone. An analysis of pregnancy rates suggests a significant treatment effect for metformin and clomifene (OR 4.40, CI 1.96 to 9.85). Metformin has a significant effect in reducing fasting insulin levels (WMD -5.37, CI -8.11 to -2.63), blood pressure and low-density lipoprotein cholesterol (LDL). There was no evidence of effect on body mass index or waist:hip ratio. Metformin was associated with a significantly higher incidence of nausea, vomiting and other gastrointestinal disturbance, but no serious adverse effects were reported.Metformin is an effective treatment for anovulation in women with PCOS. Its choice as a first line agent seems justified, and there is some evidence of benefit on parameters of the metabolic syndrome. Ovulation rates are higher when combined with clomifene (76% versus 46% when used alone), but there is no evidence to indicate whether there is an increased multiple pregnancy rate with this combination. There is no data regarding its safety in long-term use in young women. It should be used as an adjuvant to general lifestyle improvements, and not as a replacement for increased exercise and improved diet.

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-delta. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC-delta signaling pathways.

Protein kinase C-delta is activated during apoptosis, following proteolytic cleavage by caspase 3. Furthermore, overexpression of the catalytic kinase fragment of PKC-delta induces the nuclear phenotype associated with apoptosis, though the molecular basis of this effect has not been determined. In these studies we have examined the role of PKC-delta in the disassembly of the nuclear lamina at apoptosis. The nuclear lamina is disassembled during mitosis and apoptosis and mitotic disassembly involves hyperphosphorylation of lamin proteins by mitotic lamin kinases. During apoptosis, lamin proteins are degraded by caspase 6 and the contribution made by phosphorylation has not been proven. We show here that protein kinase C-delta co-localized with lamin B during apoptosis and activation of PKC-delta by caspase 3 was concomitant with lamin B phosphorylation and proteolysis. Inhibition of PKC-delta delayed lamin proteolysis, even in the presence of active caspase 6, whilst inhibitors of mitotic lamin kinases were without effect. In addition recombinant human PKC-delta was able to phosphorylate lamin B in vitro suggesting that its actions are direct and not via an intermediary kinase. We propose that PKC-delta is an apoptotic lamin kinase and that efficient lamina disassembly at apoptosis requires both lamin hyperphosphorylation and caspase mediated proteolysis.

Amongst lymphocytes, expression of CD77 (globotriaosylceramide, Gb3) is exclusive to B cells of the germinal center (GC). Its acquisition by extrafollicular B cells may thus herald their commitment to a follicular response. Here we show that high threshold occupancy of CD40 by its cognate ligand (CD40L) promotes rapid induction of CD77 expression in non-GC (CD38(lo)) B cells. The kinetics of CD77 acquisition mirrored those of GC-related markers CD95 and CD86 but contrasted with the more delayed increase in CD38 expression. Induction of CD77 was not a simple consequence of cell cycle entry: other conditions of stimulation equally capable of driving proliferation failed to promote CD77 expression. CD77 was functional in that cells were now sensitive to Verotoxin-1, an Escherichia coli-derived ligand of Gb3. These data indicate that acquisition by extrafollicular B cells of CD77 results from high threshold occupancy of CD40, a situation that should be reached physiologically only once a critical level of T cell priming has been achieved.

Type I interferons rescue activated human T cells from cytokine deprivation-induced apoptosis. Our data now show that IFN-beta also rapidly inhibits apoptotic signals induced through the Fas receptor (CD95) in human T cells. To identify upstream signaling elements that could be targets of IFN-beta, we have studied protein kinase C (PKC). PKC-delta is actively involved in the regulation of apoptosis and immunofluorescence staining revealed that early in apoptosis PKC-delta accumulated in the nucleus. Addition of IFN-beta to T cells already deprived of survival factors or treated with anti-Fas antibody caused a rapid retranslocation of PKC-delta away from the nucleus. Furthermore, the generation of a constitutively active catalytic fragment by cleavage of PKC-delta by caspase 3 occurred only after translocation of full-length PKC-delta to the nucleus. IFN-beta also inhibited caspase 3 and the proteolytic activation of PKC-delta. We conclude from these studies that nuclear translocation of PKC-delta is an early event in T cell apoptosis and that IFN-beta rapidly reverses this process.

Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin-a, abrin-b, abrin-c and abrin-d), whose B chains are believed to either have (abrin-a and abrin-d) or lack (abrin-b and abrin-c) the ability to bind galactose. The 5' signal sequence and toxin B chain (ATB)-coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem. 198, 723-732], tentatively identified as abrin-c, which was predicted to lack lectin activity, and fused in-frame to generate pre-ATB cDNA. Transcripts, synthesized in vitro from pre-ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar-binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2 gamma subdomain with the corresponding galactose-binding 2 gamma subdomain from ricin B chain, but it was restored by replacement with the active galactose-binding 2 gamma subdomain from a different abrin isoform (abrin-a). The putative galactose-binding pocket of the nonbinding ATB 2 gamma subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2 gamma subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2 gamma subdomain galactose-binding site for members of this lectin family.

Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.

Newly synthesized proteins that fail to fold or assemble properly in the endoplasmic reticulum are degraded. Recent work on several endoplasmic reticulum membrane proteins has shown that the cytosolic proteasome plays a role in their degradation.

Recent reports have claimed that activation of protein kinase C (PKC)-beta is sufficient for both differentiation and apoptosis in promyeloid HL60 cells. Phorbol esters which differentially activate PKC isoenzymes in vitro were used to induce differentiation and apoptosis in U937 cells; TPA and Dopp activate all U937 PKC isoenzymes, except PKC-zeta and Doppa activate only PKC-beta l. At concentrations of Doppa below 50 nM, only PKC-beta l was activated by 2 min and apoptosis was induced, but there was no differentiation of cells towards monocytes. TPA (1-25 nM) and Dopp (5-100 nM) activated PKC-alpha, -beta l and-gamma within 2 min and induced differentiation, but only increased apoptosis at the highest concentrations used. Thus, initial activation of PKC-beta l is insufficient for differentiation of U937 cells, but may lead to the induction of apoptosis.

Following cellular uptake, Pseudomonas exotoxin (PE) is cleaved by cellular protease which generates an enzymatically active C-terminal fragment (amino acids 280-613). This 37 kD fragment translocates to the cell cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. A recombinant hybrid toxin (designated PE-RTA) in which the ADP-ribosylation domain (domain 111) was replaced by the RNA N-glycosidase domain of ricin (the A chain or RTA) has been produced in E. coli. The hybrid toxin effectively and specifically depurinated 28S ribosomal RNA, indicating that the ricin A moiety folded into its native conformation. The cytotoxicity of PE-RTA for L929 cells was approximately 100-fold less than either native PE or whole ricin. However, the addition of the tetrapeptide KDEL to the C-terminus of PE-RTA (producing PE-RTA KDEL) increased cytotoxicity to the level of the native toxins. By analogy to PE, both PE-RTA and PE-RTA KDEL would be proteolytically cleaved within PE domain II during cell entry. A single amino acid substitution, believed to disrupt an essential step in the transport of the catalytically active PE fragment to the cell cytosol (Trp281 to Ala: Zdanovsky, A.G., Chiron, M., Pastan, I., and FitzGerald, D. J. (1993) J. Biol. Chem. 268, 21791-21799), reduced the cytotoxicities of both PE and PE-RTA KDEL by approximately 100-fold. Taken together, these data show that the ricin A chain component of the hybrid toxin requires essential PE-derived sequences at both the N- and C-termini of the translocating fragment. Clearly, in the context of this fusion protein, ricin A chain cannot effect its own transfer to the cytosol.

Ricin B-chain (RTB) is a galactose-specific lectin that folds into two globular domains, each of which binds a single galactoside. The two binding sites are structurally similar and both contain a conserved tripeptide kink and an aromatic residue that comprises a sugar-binding platform. Whereas the critical RTB residues implicated in lectin activity are conserved in domain 1 of Ricinus communis agglutinin (RCA) B-chain, the sugar platform aromatic residue Tyr-248 present in domain 2 of RTB is replaced by His in RCA B-chain. In this study, key residues in the vicinity of the binding sites of the Ricinus lectin B-chains were altered by site-directed mutagenesis. The recombinant B-chains were produced in Xenopus oocytes in soluble, stable, and core-glycosylated forms. Both sites of RCA B-chain must be simultaneously modified in order to abolish lectin activity, indicating the presence of two independent, functional binding sites/molecule. Activity associated with the domain 2 site of RCA B-chain is abrogated by the conversion of Trp-258 to Ser. Moreover, the domain 2 site appears responsible for a weak binding interaction recombinant RCA B-chain with GalNAc, not observed with native tetrameric RCA. Finally, the introduction of His at position 248 of RTB severely disrupts but does not abolish GalNAc binding.

Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

An Escherichia coli expression system was used to produce recombinant ricin A chain (RTA) and RTA modified either by the addition of a carboxyl-terminal endoplasmic reticulum retrieval sequence Lys-Asp-Glu-Leu (RTAKDEL) or a nonfunctional analogue Lys-Asp-Glu-Ala (RTAKDEA). These RTA molecules can enter mammalian cells by fluid phase endocytosis. RTAKDEL was significantly more cytotoxic than either RTA or RTAKDEA to both Vero cells and HeLa cells (250- and 10-fold, respectively), despite the fact that all these RTA molecules had comparable enzymatic activities. This difference did not result from KDEL-mediated binding of RTAKDEL to the cell surface. Enhanced cytotoxicity could be correlated with an increased level of ribosome inactivation, measured as the RTA-catalyzed depurination of 28 S ribosomal RNA. These results indicate that the added KDEL sequence facilitated RTA entry into the cytosol. We propose that interaction with the intracellular KDEL receptor promotes retrograde transport of the toxin to the endoplasmic reticulum, where translocation of RTA into the cytosol occurs.

Ricin is the best known example of a family of proteins known as ribosome-inactivating proteins or RIPs. These are widely distributed in Angiosperms and also occur in several species of fungi and bacteria. A commonly used criterion for the inclusion of proteins within this group is that they catalytically and irreversibly inactivate the 60S subunit of eukaryotic ribosomes, rendering it incapable of binding elongation factor 2 (Stirpe and Barbieri, 1986). Although this definition was proposed before the nature of the RIP-mediated modifications to ribosomes were known, it remains a useful working definition but should be broadened to take into account the recent finding that some RIPs are also active on prokaryotic ribosomes. Protein toxins which act by the catalytic inactivation of protein synthesis accessory factors such as diphtheria toxin and Pseudomonas exotoxin are not included within this group.

The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990) FEBS Lett. 273,200. Kim, J., and Weaver, R.F. (1988) Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosome-inactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavage site in recombinant fusions with RTA is discussed.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.

A variety of strategies have been used to obtain cDNA and genomic clones encoding ricin. Since their isolation these sequences have been manipulated to allow expression of A chain (19) and A chain mutants (15,20,34), B chain (14,21-23) and proricin (24). Utilizing structural information (35), precise changes have been introduced into both A and B chains with the aim of probing catalytic and sugar-binding residues, respectively. In the longer term, such manipulations, coupled with successful expression and purification schemes, will allow the delineation of functional residues and domains, ensuring that ricin remains the prototype plant toxin with which to study cellular intoxication and ribosome inactivation and to utilize in pharmaceutical product development.

Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.

Many plant tissues produce single chain proteins which can enzymatically remove a specific adenine residue from ribosomal RNA. Although these proteins are potently toxic to isolated ribosomes, they are non-toxic to intact cells, being unable to gain access to their ribosomal substrate. In certain plants however, the gene for the ribosome inactivating protein has fused with a gene encoding a galactose-specific lectin. This generates heterodimeric proteins which can bind to and enter target cells, and which are among the most potent cytotoxins known.

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of mannose from GDP[(14)C]mannose into acid-labile mannolipids. Solubility and chromatographic properties have identified the most rapidly synthesized products as mannosyl-phosphoryl-polyisoprenol, while the more polar lipid formed was shown to contain oligosaccharide. Little radioactivity from GDP[(14)C]mannose accumulated in insoluble product in the cell-free system, but supplying GDP[(14)C]mannose to intact endosperm tissue has shown that the major incorporation product in vivo is glycoprotein. This product was readily solubilized by either pronase or sodium dodecyl sulphate treatment suggesting it was membrane bound glycoprotein. Incorporation of mannose into mannosyl-phosphoryl-polyisoprenol during the cell-free assay was stimulated by the addition of dolichol monophosphate. This enzymic activity was optimal at pH 7.5 and in the presence of 10 mM Mg(2+). The Km for GDP-mannose was estimated to be 5×10(-7) M. Cellular mannosyl transferase activity changed markedly during early post-germinative growth; from being absent in the dry seed, enzyme activity increased to peak between the second and third days of growth and subsequently declined.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of galactose from UDP[(14)C]galactose into chloroform/methanol (2:1)-soluble glactolipids. At least two galactolipids were formed. Most of the [(14)C]galactose was present in a galactolipid synthesized by the microsomal membranes, the remainder was present in a second galactolipid synthesized by other cellular membranes, possibly Golgi-derived. The addition of asialo-agalacto-fetuin reduced incorporation of [(14)C]galactose into the microsomal galactolipid with a concomitant increase in microsomal [(14)C]galactoprotein. Asialo-agalacto-fetuin did not affect galactolipid or galactoprotein synthesis by nonmicrosomal fractions. The results suggest that the endoplasmic reticulum is a major site of protein galactosylation in castor bean endosperm cells, and that galactose transfer from UDP-galactose to protein occurs via a lipid-linked intermediate.

Ecological diversity provides a means of analysing the community structure of fossil mammalian faunas in order to obtain information on the habitat of the fauna. As a basis for the analysis, 23 modern mammalian communities from distinct habitats have been used to establish patterns of community structure for tropical African habitats according to their species diversity by taxonomic group, size, locomotor zonal adaptation, and feeding adaptation. All the communities tested were in tropical Africa, but additional analyses on tropical forest communities in Australia, Malaya, and Panama have shown that these communities, which all have completely different species composition, nevertheless have community structures very similar to each other and to those of the African forest communities.

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [(35)S]methionine and the kinetics of (35)S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [(35)S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [(35)S]antigens and a concomitant increase in glyoxysomal content.

Lectins are a group of proteins and glycoproteins which possess specific binding sites for particular sugars. They are very abundant in certain plant tissues, for example the seeds of legumes. Although their biological role in plants has not been convincingly explained, details of their structure, synthesis and molecular biology are beginning to emerge. Contents Summary 351 I. Introduction 351 II. Structure of lectins 352 III. Synthesis of lectins 359 IV. Conclusion 363 References 363.

INTRODUCTION . . . . 751 GLYCOLLATE FORMATION DURING PHOTOSYNTHESIS. 753 GLYCOLLATE EXCRETION BY ALGAE . 755 REGULATION OF GLYCOLLATE EXCRETION BY ALGAE. . 755 EFFECT OF LIGHT QUALITY ON GLYCOLLATE BIOSYNTHESIS BY ALGAE. . . . 757 GLYCOLLATE OXIDIZING ENZYME OF ALGAE. 757 REGULATION OF GLYCOLLATE DEHYDROGENASE ACTIVITY IN ALGAE . 758 GLYCOLLATE PATHWAY IN ALGAE 758 PHOTOHETEROTROPHIC GROWTH OF ALGAE ON GLYCOLLATE . \. . 760 INTRACELLULAR LOCALIZATION OF THE GLYCOLLATE PATHWAY IN ALGAE . 760 PHOTORESPIRATION IN ALGAE . . . . . . . . . 762 CONCLUSIONS . . . . . . . . . . . 763 REFERENCES . . . . . . . . . 764

The intracellular location of several enzymes concerned with phospholipid metabolism was investigated by examining their distribution in organelles separated on sucrose gradients from total homogenates of castor bean (Ricinus communis var. Hale) endosperm. The enzymes phosphatidic acid phosphatase, CDP-diglyceride-inositol transferase, and phosphatidyletha-nolamine-l-serine phosphatidyl transferase were all primarily or exclusively confined to membranes of the endoplasmic reticulum. These results and those reported previously on lecithin synthesis establish a major role of the endoplasmic reticulum in phospholipid and membrane synthesis in plant tissues.