Uncoupling DNA damage from chromatin damage to detoxify doxorubicin.

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.

Synthetic (N,N-Dimethyl)doxorubicin Glycosyl Diastereomers to Dissect Modes of Action of Anthracycline Anticancer Drugs.

Anthracyclines are effective drugs in the treatment of various cancers, but their use comes with severe side effects. The archetypal anthracycline drug, doxorubicin, displays two molecular modes of action: DNA double-strand break formation (through topoisomerase IIα poisoning) and chromatin damage (via eviction of histones). These biological activities can be modulated and toxic side effects can be reduced by separating these two modes of action through alteration of the aminoglycoside moiety of doxorubicin. We herein report on the design, synthesis, and evaluation of a coherent set of configurational doxorubicin analogues featuring all possible stereoisomers of the 1,2-amino-alcohol characteristic for the doxorubicin 3-amino-2,3-dideoxyfucoside, each in nonsubstituted and N,N-dimethylated forms. The set of doxorubicin analogues was synthesized using appropriately protected 2,3,6-dideoxy-3-amino glycosyl donors, equipped with an alkynylbenzoate anomeric leaving group, and the doxorubicin aglycon acceptor. The majority of these glycosylations proceeded in a highly stereoselective manner to provide the desired axial α-linkage. We show that both stereochemistry of the 3-amine carbon and N-substitution state are critical for anthracycline cytotoxicity and generally improve cellular uptake. N,N-Dimethylepirubicin is identified as the most potent anthracycline that does not induce DNA damage while remaining cytotoxic.

Immunoproteasome Inhibitor-Doxorubicin Conjugates Target Multiple Myeloma Cells and Release Doxorubicin upon Low-Dose Photon Irradiation.

Proteasome inhibitors are established therapeutic agents for the treatment of hematological cancers, as are anthracyclines such as doxorubicin. We here present a new drug targeting approach that combines both drug classes into a single molecule. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active toward immunoproteasomes. Upon cellular uptake and immunoproteasome inhibition, doxorubicin is released from the immunoproteasome inhibitor through photoirradiation. Multiple myeloma cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity. Our strategy, which entails targeting of a cytotoxic agent, through a covalent enzyme inhibitor that is detrimental to tumor tissue in its own right, may find use in the search for improved anticancer drugs.

Outcomes with durvalumab by tumour PD-L1 expression in unresectable, stage III non-small-cell lung cancer in the PACIFIC trial.

BACKGROUND: In the PACIFIC trial, durvalumab significantly improved progression-free and overall survival (PFS/OS) versus placebo, with manageable safety, in unresectable, stage III non-small-cell lung cancer (NSCLC) patients without progression after chemoradiotherapy (CRT). We report exploratory analyses of outcomes by tumour cell (TC) programmed death-ligand 1 (PD-L1) expression. PATIENTS AND METHODS: Patients were randomly assigned (2:1) to intravenous durvalumab 10 mg/kg every 2 weeks or placebo ≤12 months, stratified by age, sex, and smoking history, but not PD-L1 status. Where available, pre-CRT samples were tested for PD-L1 expression (immunohistochemistry) and scored at pre-specified (25%) and post hoc (1%) TC cut-offs. Treatment-effect hazard ratios (HRs) were estimated from unstratified Cox proportional hazards models (Kaplan-Meier-estimated medians). RESULTS: In total, 713 patients were randomly assigned, 709 of whom received at least 1 dose of study treatment durvalumab (n = 473) or placebo (n = 236). Some 451 (63%) were PD-L1-assessable: 35%, 65%, 67%, 33%, and 32% had TC ≥25%, <25%, ≥1%, <1%, and 1%-24%, respectively. As of 31 January 2019, median follow-up was 33.3 months. Durvalumab improved PFS versus placebo (primary-analysis data cut-off, 13 February 2017) across all subgroups [HR, 95% confidence interval (CI); medians]: TC ≥25% (0.41, 0.26-0.65; 17.8 versus 3.7 months), <25% (0.59, 0.43-0.82; 16.9 versus 6.9 months), ≥1% (0.46, 0.33-0.64; 17.8 versus 5.6 months), <1% (0.73, 0.48-1.11; 10.7 versus 5.6 months), 1%-24% [0.49, 0.30-0.80; not reached (NR) versus 9.0 months], and unknown (0.59, 0.42-0.83; 14.0 versus 6.4 months). Durvalumab improved OS across most subgroups (31 January 2019 data cut-off; HR, 95% CI; medians): TC ≥ 25% (0.50, 0.30-0.83; NR versus 21.1 months), <25% (0.89, 0.63-1.25; 39.7 versus 37.4 months), ≥1% (0.59, 0.41-0.83; NR versus 29.6 months), 1%-24% (0.67, 0.41-1.10; 43.3 versus 30.5 months), and unknown (0.60, 0.43-0.84; 44.2 versus 23.5 months), but not <1% (1.14, 0.71-1.84; 33.1 versus 45.6 months). Safety was similar across subgroups. CONCLUSIONS: PFS benefit with durvalumab was observed across all subgroups, and OS benefit across all but TC <1%, for which limitations and wide HR CI preclude robust conclusions.

Carba-cyclophellitols Are Neutral Retaining-Glucosidase Inhibitors.

The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine-both covalent retaining ß-glucosidase inhibitors-we postulated that the corresponding carba "cyclopropyl" analogue would be a potent retaining ß-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of the Thermotoga maritima TmGH1 ß-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.

Metabolic engineering of Streptomyces peucetius for biosynthesis of N,N-dimethylated anthracyclines.

Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius, are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N-dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N-dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001. To achieve this, we introduced genes from the aclarubicin biosynthetic pathway encoding the sugar N-methyltransferases AclP and AknX2. Furthermore, the native gene for glycosyltransferase DnrS was replaced with genes encoding the aclarubicin glycosyltransferases AknS and AknT. Additionally, the gene for methylesterase RdmC from the rhodomycin biosynthetic pathway was introduced. Results: A new host was engineered successfully, whereby genes from the aclarubicin pathway were introduced and expressed. LC-MS/MS analysis of the engineered strains showed that dimethylated sugars were efficiently produced, and that these were incorporated ino the anthracycline biosynthetic pathway to produce the novel dimethylated anthracycline N,N-dimethyldaunorubicin. Further downstream tailoring steps catalysed by the cytochrome P450 monooxygenase DoxA exhibited limited efficacy with N,N-dimethylated substrates. This resulted in only low production levels of N,N-dimethyldaunorubicin and no N,N-dimethyldoxorubicin, most likely due to the low affinity of DoxA for dimethylated substrates. Discussion: S. peucetius G001 was engineered such as to produce N,N-dimethylated sugars, which were incorporated into the biosynthetic pathway. This allowed the successful production of N,N-dimethyldaunorubicin, an anticancer drug with reduced cytotoxicity. DoxA is the key enzyme that determines the efficiency of the biosynthesis of N,N-dimethylated anthracyclines, and engineering of this enzyme will be a major step forwards towards the efficient production of more N,N-dimethylated anthracyclines, including N,N-dimethyldoxorubicin. This study provides valuable insights into the biosynthesis of clinically relevant daunorubicin derivatives, highlighting the importance of combinatorial biosynthesis.

Re-Exploring the Anthracycline Chemical Space for Better Anti-Cancer Compounds.

The anthracycline anti-cancer drugs are intensely used in the clinic to treat a wide variety of cancers. They generate DNA double strand breaks, but recently the induction of chromatin damage was introduced as another major determinant of anti-cancer activity. The combination of these two events results in their reported side effects. While our knowledge on the structure-activity relationship of anthracyclines has improved, many structural variations remain poorly explored. Therefore, we here report on the preparation of a diverse set of anthracyclines with variations within the sugar moiety, amine alkylation pattern, saccharide chain and aglycone. We assessed the cytotoxicity in vitro in relevant human cancer cell lines, and the capacity to induce DNA- and chromatin damage. This coherent set of data allowed us to deduce a few guidelines on anthracycline design, as well as discover novel, highly potent anthracyclines that may be better tolerated by patients.

Outcomes with durvalumab after chemoradiotherapy in stage IIIA-N2 non-small-cell lung cancer: an exploratory analysis from the PACIFIC trial.

BACKGROUND: The phase III PACIFIC trial (NCT02125461) established consolidation durvalumab as standard of care for patients with unresectable, stage III non-small-cell lung cancer (NSCLC) and no disease progression following chemoradiotherapy (CRT). In some cases, patients with stage IIIA-N2 NSCLC are considered operable, but the relative benefit of surgery is unclear. We report a post hoc, exploratory analysis of clinical outcomes in the PACIFIC trial, in patients with or without stage IIIA-N2 NSCLC. MATERIALS AND METHODS: Patients with unresectable, stage III NSCLC and no disease progression after ≥2 cycles of platinum-based, concurrent CRT were randomized 2 : 1 to receive durvalumab (10 mg/kg intravenously; once every 2 weeks for up to 12 months) or placebo, 1-42 days after CRT. The primary endpoints were progression-free survival (PFS; assessed by blinded independent central review according to RECIST version 1.1) and overall survival (OS). Treatment effects within subgroups were estimated by hazard ratios (HRs) from unstratified Cox proportional hazards models. RESULTS: Of 713 randomized patients, 287 (40%) had stage IIIA-N2 disease. Baseline characteristics were similar between patients with and without stage IIIA-N2 NSCLC. With a median follow-up of 14.5 months (range: 0.2-29.9 months), PFS was improved with durvalumab versus placebo in both patients with [HR = 0.46; 95% confidence interval (CI), 0.33-0.65] and without (HR = 0.62; 95% CI 0.48-0.80) stage IIIA-N2 disease. Similarly, with a median follow-up of 25.2 months (range: 0.2-43.1 months), OS was improved with durvalumab versus placebo in patients with (HR = 0.56; 95% CI 0.39-0.79) or without (HR = 0.78; 95% CI 0.57-1.06) stage IIIA-N2 disease. Durvalumab had a manageable safety profile irrespective of stage IIIA-N2 status. CONCLUSIONS: Consistent with the intent-to-treat population, treatment benefits with durvalumab were confirmed in patients with stage IIIA-N2, unresectable NSCLC. Prospective studies are needed to determine the optimal treatment approach for patients who are deemed operable.

Regional implementation of atrial fibrillation screening: benefits and pitfalls.

Aims: Despite general awareness that screening for atrial fibrillation (AF) could reduce health hazards, large-scale implementation is lagging behind technological developments. As the successful implementation of a screening programme remains challenging, this study aims to identify facilitating and inhibiting factors from healthcare providers' perspectives. Methods and results: A mixed-methods approach was used to gather data among practice nurses in primary care in the southern region of the Netherlands to evaluate the implementation of an ongoing single-lead electrocardiogram (ECG)-based AF screening programme. Potential facilitating and inhibiting factors were evaluated using online questionnaires (N = 74/75%) and 14 (of 24) semi-structured in-depth interviews (58.3%). All analyses were performed using SPSS 26.0. In total, 16 682 screenings were performed on an eligible population of 64 000, and 100 new AF cases were detected. Facilitating factors included 'receiving clear instructions' (mean ± SD; 4.12 ± 1.05), 'easy use of the ECG-based device' (4.58 ± 0.68), and 'patient satisfaction' (4.22 ± 0.65). Inhibiting factors were 'time availability' (3.20 ± 1.10), 'insufficient feedback to the practice nurse' (2.15 ± 0.89), 'absence of coordination' (54%), and the 'lack of fitting policy' (32%). Conclusion: Large-scale regional implementation of an AF screening programme in primary care resulted in a low participation of all eligible patients. Based on the perceived barriers by healthcare providers, future AF screening programmes should create preconditions to fit the intervention into daily routines, appointing an overall project lead and a General Practitioner (GP) as a coordinator within every GP practice.

Doxorubicin and Aclarubicin: Shuffling Anthracycline Glycans for Improved Anticancer Agents.

Anthracycline anticancer drugs doxorubicin and aclarubicin have been used in the clinic for several decades to treat various cancers. Although closely related structures, their molecular mode of action diverges, which is reflected in their biological activity profile. For a better understanding of the structure-function relationship of these drugs, we synthesized ten doxorubicin/aclarubicin hybrids varying in three distinct features: aglycon, glycan, and amine substitution pattern. We continued to evaluate their capacity to induce DNA breaks, histone eviction, and relocated topoisomerase IIα in living cells. Furthermore, we assessed their cytotoxicity in various human tumor cell lines. Our findings underscore that histone eviction alone, rather than DNA breaks, contributes strongly to the overall cytotoxicity of anthracyclines, and structures containing N,N-dimethylamine at the reducing sugar prove that are more cytotoxic than their nonmethylated counterparts. This structural information will support further development of novel anthracycline variants with improved anticancer activity.

Defining the SN1 Side of Glycosylation Reactions: Stereoselectivity of Glycopyranosyl Cations.

The broad application of well-defined synthetic oligosaccharides in glycobiology and glycobiotechnology is largely hampered by the lack of sufficient amounts of synthetic carbohydrate specimens. Insufficient knowledge of the glycosylation reaction mechanism thwarts the routine assembly of these materials. Glycosyl cations are key reactive intermediates in the glycosylation reaction, but their high reactivity and fleeting nature have precluded the determination of clear structure-reactivity-stereoselectivity principles for these species. We report a combined experimental and computational method that connects the stereoselectivity of oxocarbenium ions to the full ensemble of conformations these species can adopt, mapped in conformational energy landscapes (CEL), in a quantitative manner. The detailed description of stereoselective SN1-type glycosylation reactions firmly establishes glycosyl cations as true reaction intermediates and will enable the generation of new stereoselective glycosylation methodology.

Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation.

To evaluate the role of Akt/PKB in non-small cell lung cancer (NSCLC) survival, we analyzed NSCLC cell lines that differed in tumor histology as well as p53, Rb, and K-ras status. Constitutive Akt/protein kinase B (PKB) activity was demonstrated in 16 of 17 cell lines by maintenance of S473 phosphorylation with serum deprivation. Additional analysis of five of 2these NSCLC lines revealed that phosphorylation of S473 and T308 correlated with in vitro kinase activity. Akt/PKB activation was phosphatidylinositol 3-kinase-dependent and promoted survival because the phosphatidylinositol 3 inhibitors LY294002 and wortmannin inhibited Akt/PKB phosphorylation, Akt/PKB activity, and increased apoptosis only in cells with active Akt/PKB. To test whether Akt/PKB activity promoted therapeutic resistance, LY294002 was added with individual chemotherapeutic agents or irradiation. LY294002 greatly potentiated chemotherapy-induced apoptosis in cells with high Akt/PKB levels, but did not significantly increase chemotherapy-induced apoptosis in cells with low Akt/PKB levels. Combined with radiation in cells with active Akt/PKB, LY294002 additively increased apoptosis and inhibited clonogenic growth. These results were extended with transiently transfected Akt/PKB mutants. Transfecting dominant negative Akt/PKB decreased Akt/PKB activity and increased basal apoptosis as well as chemotherapy- and irradiation-induced apoptosis only in cells with high Akt/PKB activity. Conversely, transfecting constitutively active Akt/PKB into cells with low Akt/PKB activity increased Akt/PKB activity and attenuated chemotherapy- and radiation-induced apoptosis. We therefore identify Akt/PKB as a constitutively active kinase that promotes survival of NSCLC cells and demonstrate that modulation of Akt/PKB activity by pharmacological or genetic approaches alters the cellular responsiveness to therapeutic modalities typically used to treat patients with NSCLC.

Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2.

We investigated whether combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and trastuzumab could enhance the specific killing of cells that overexpress the erbB-2 receptor. The combination resulted in an enhancement of TRAIL-mediated apoptosis in all cell lines overexpressing erbB-2 receptor compared with either reagent alone. In contrast, there was no effect in cell lines with low levels of the erb-B2 receptor. Trastuzumab treatment resulted in down-regulation of the erbB-2 receptor in all erbB-2-overexpressing cell lines. Similar enhancement of TRAIL toxicity was observed when the erbB-2 receptor was down-regulated using antisense oligodeoxynucleotides. Down-regulation of the erbB-2 receptor protein by trastuzumab or antisense oligodeoxynucleotides decreased Akt kinase activation but not mitogen-activated protein kinase activation. Down-regulation of Akt kinase activity by a phosphatidylinositol 3'-kinase inhibitor (LY294002) also resulted in enhancement of TRAIL-mediated apoptosis. Expression of a constitutively active form of Akt kinase in an erbB-2-overexpressing cell line completely abrogated the increase in TRAIL-mediated apoptosis by trastuzumab and significantly reduced the biological effect of either reagent alone. Therefore, down-regulation of the erbB-2 receptor by trastuzumab enhances TRAIL-mediated apoptosis by inhibiting Akt kinase activity. These data suggest that the combination of trastuzumab and TRAIL may allow enhanced therapeutic efficacy and specificity in the treatment of erbB-2-overexpressing tumors.

Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.

Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.

Uptake and degradation of 125I-labeled rat asialoorosomucoid by the perfused rat liver.

The uptake and degradation of a homologous rat serum asialoglycoprotein, 125I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. 125I-Asialoorosomucoid was rapidly taken up by the liver (t1/2 = 5.7 min) and acid-soluble degradation products began to appear in the circulating perfusate medium after 20-30 min. These products accounted for 60-65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of 125I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of 125I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathepsin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat 125I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occurred in the native molecular weight of the starting glycoprotein.

Variable apoptotic response of NSCLC cells to inhibition of the MEK/ERK pathway by small molecules or dominant negative mutants.

To evaluate the role of the MEK/ERK pathway in NSCLC survival, we analyzed NSCLC cell lines that differed in tumor histology and status of p53, Rb, and K-ras. Constitutive ERK1/2 activity was demonstrated in 17 of 19 cell lines by maintenance of ERK1/2 phosphorylation with serum deprivation. Phosphorylation of ERK1/2 correlated with phosphorylation of MEK1/2 and p90RSK, but was inversely correlated with phosphorylation of c-Raf at S259. With serum deprivation, the MEK inhibitors, PD98059 and U0126, inhibited ERK1/2 activity but did not increase apoptosis. PD98059 and U0126 induced cell cycle arrest in G(0)/G(i) in cells with the highest levels of ERK1/2 activity, which correlated with induction of p27 but not p21. To confirm the cytostatic response to MEK inhibitors, we performed transient transfections with dominant negative forms of MEK or ERK. Surprisingly, dominant negative MEK and ERK mutants increased apoptosis without affecting cell cycle or p27 levels. When combined with paclitaxel, MEK inhibitors had no effect on apoptosis. In contrast, dominant negative ERK2 potentiated paclitaxel-induced apoptosis. Our studies show that constitutive ERK1/2 activity in NSCLC cells promotes cellular survival and chemotherapeutic resistance. Moreover, our data are the first to demonstrate divergent cellular responses to inhibition of the MEK/ERK pathway by small molecule inhibitors or dominant negative mutants.

Hemodialysis without anticoagulation. One-year prospective trial in hospitalized patients at risk for bleeding.

This prospective study evaluated a protocol for hemodialysis without anticoagulation in a diverse group of hospitalized patients in unstable condition with relative contraindications to anticoagulation. Of 262 attempts at hemodialysis without anticoagulation in 49 patients, 239 hemodialysis treatments (91 percent) were successfully completed. Approximately 7 percent of the attempts required conversion to a low-dose heparin regimen because of clotting in the extracorporeal dialysis circuit. Fewer than 2 percent of the dialysis treatments resulted in clotting in the extracorporeal circuit sufficient to interrupt hemodialysis. Partial thromboplastin times and activated clotting times did not change during these hemodialysis treatments. Solute clearance, ultrafiltration rate, and decrements in arterial oxygen concentration and platelet count were not different from those in patients who underwent hemodialysis with anticoagulation. There were no episodes of accelerated bleeding associated with this dialysis method. This study indicates that hemodialysis without anticoagulation can be reliable and effective in closely monitored situations.

Identification of insecticidal peptides from venom of the trap-door spider, Aptostichus schlingeri (Ctenizidae).

Nine insecticidal peptides were isolated from the venom of the trap-door spider, Aptostichus schlingeri. Seven of these toxins cause flaccid paralysis of insect larvae within 10 min of injection and all were lethal within 24 hr. The complete amino acid sequences (32-76 residues) of six peptides are presented. The identified peptides contain three or four disulfide bonds and the larger peptides (74-76 residues) are quite similar in sequence with carboxyl termini as free acids, not amidated.

Chemical characterization of acylpolyamine toxins from venom of a trap-door spider and two tarantulas.

Two acylpolyamines are identified from venom of the trap-door spider, Hebestatis theveniti. These toxins (paralytic to lepidopteran insect larvae) are amides containing 3-(3-indolyl)lactic acid joined to spermine or 1,13-diamino-4,10-diazatridecane (Het389 and Het403, respectively). Het389 is also abundant in venom from a tarantual from Mozambique (Harpactirella sp.). Two additional acylpolyamines (Apc600 and Apc728) are partially characterized from venom of another tarantula, Aphonopelma chalcodes.

Paralytic and insecticidal toxins from the funnel web spider, Hololena curta.

One peptide and ten acylpolyamine toxins (curtatoxins) were purified and identified from venom of Hololena curta. The acylpolyamines consist of six different polyamines which are amidated with three different aromatic acids: (3-indolyl)acetic, (4-hydroxy-3-indolyl)acetic and 2.5-dihydroxybenzoic acids. These acylpolyamines instantly paralyze lepidopteran larvae following injection. The most potent insecticidal peptide in H. curta venom contains 38 amino acids and is lethal at 4 micrograms/g when injected into Manduca sexta larvae.