CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients

Purpose: Many years ago, diphtheria toxin (DT) showed antitumor activity in mice and in humans, but it was unclear whether this depended on the toxicity of the molecule only or on its strong inflammatory-immunological property as well. To deal with this open question, we planned to treat a group of cancer patients with cross-reacting material 197 (CRM197). CRM197 is a nontoxic mutant of DT that shares the immunological properties of the native molecule and its ability to bind to heparin-binding epidermal growth factor (HB-EGF), the specific cell-membrane receptor for DT that is often overexpressed in cancer. Methods: 25 outpatients with various advanced tumors who were refractory to standard therapies (23 subjects) or had refused, in whole or in part, conventional therapies (2 subjects) were treated with CRM197 injected subcutaneously in the abdominal wall, on alternate days, for 6 days. Three different dosages (1.7, 2.6, or 3.5 mg/day) were used according to the patients degree of immunological reactivity to DT/CRM197 (none, moderate, or high). Results: After the first administration of CRM197, a significant increase in the number of circulating neutrophils and in the serum level of TNF- was detected. Toxicities were minimal. Only patients with delayed-type hypersensitivity to DT/CRM197 had irritating skin reactions in the injection sites and a flu-like syndrome with fever. Pharmacokinetics showed a mean peak concentration (12.7 ng/ml) 12 h after the first injection and a mean half-life of 18.1 h. There were two complete and one partial responses (metastatic breast carcinoma, neuroblastoma, and metastatic breast carcinoma) lasting 4, 45+, and 15 months, respectively. Six cases of stable disease, lasting from 1 to 15 months, were also recorded. Conclusions: CRM197 injected subcutaneously elicited an inflammatory-immunological reaction, caused tolerable toxicities, was absorbed to a good extent into the circulatory system, and exerted some degree of biological antitumor activity. A possible role of neutrophils and TNF- in the mode of action of the molecule is hypothesized.     

Phase-synchronization of daily motor activities can reveal differential circadian patterns

The aim of the study was to determine any alteration of the 24 h motor activity pattern of a bipolar patient in different mood states. Actigraphic records were collected on an outpatient basis for a total of 387 days. The daily actograms were synchronized in phase to the time of morning awakening before averaging, which significantly enhanced the structure of the averaged traces. The actograms were divided into three groups based on total daily count. The daily motor activity patterns of the low- and high-activity days have a different circadian pattern. We propose it may have a relevance to the different mood states. The phase-synchronization of the 24 h actograms to the patient's sleep-wake cycle, specifically to the time of awaking from the nighttime sleep, may help reveal differences in the daily temporal patterns of motor activity.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

Determination of the absolute configuration of Anisotome irregular diterpenes: application of CD and NMR methods

Several Anisotome diterpene derivatives were synthesized in an attempt to obtain a crystalline compound for X-ray analysis. Although we were unable to obtain a suitable crystal, the absolute configuration of the irregular diterpene skeleton was determined using two other techniques: a circular dichroism (CD) protocol based on a tetraarylporphyrin molecular tweezer that allowed prediction of the absolute stereochemistry on a microscale level, and a method employing differences in NMR shifts from derivatization of the naturally occurring acid 1 with enantiomers of a phenylglycine methyl ester (PGME) chiral anisotropic reagent. The excellent agreement between the CD and NMR methods led to the assignment of a 2S-absolute configuration for anisotomenoic acid 1.     

The oil of Adenanthera pavonina L. seeds and its emulsions

The oil of Adenanthera pavonina L. seeds was analysed by chromatographic and instrumental means. The oil was found to be rich in neutral lipids (86.2%), and low in polar lipids (13.8%). The neutral lipids consisted mainly of triacylglycerols (64.2%). Unsaturated fatty acids were found as high as 71%, while the percentage of saturated fatty acids was only 29%. GC and GC/MS analyses revealed linoleic, oleic and lignocerotic acid to be predominant among all fatty acids in the A. pavonina oil, whereas stigmasterol was the major steroid identified within this study. Subsequently, the oil was used for preparation of submicron oil-in-water (o/w) lipid emulsions. Lipid emulsions were formulated by using soybean lecithin (SL) to investigate their particle size, Zeta potential and stability at the different oil and SL ratios. The results obtained indicate possible applications of the tested oil in pharmaceutical and medical fields as drug and cosmetic active ingredient carriers.     

Comparative LCA of industrial objects

Goal, Scope and Background  This paper is the second part of the publication which is devoted to comparative LCA analysis of the industrial pumps. The previous paper deals with the methodological aspects concerning quality assessment and forms an independent work. This paper uses practically only the methodological suggestions made there. The main aim of the presented study is to make a comparison between the industrial pumps which are based on two different technologies. The Life Cycle Assessment method is used to check whether the differences of the manufacturing processes influence the level of the potential environmental impact during the whole life cycle of the analysed products. Methods  The Life Cycle Assessment is carried out using the Ecoindicator99 method. Additionally, an extensive quality analysis of the LCA study is made (Part I). To make the process of an identification of the data easier and faster, they are assigned to a special data documentation form. To ensure the credibility of the LCA results different methods of interpretation are used. Results and Discussion  The LCA analysis shows clear superiority of the pumps manufactured using modern technology. It seems that this superiority results not only from the differences in the emissions, but also from different characteristics of effectiveness in the usage stage. Thanks to the uncertainty analysis, each LCA result is provided with the range of uncertainty. Conclusions  The LCA results are supported by different techniques of interpretation: the sensitivity-, the contribution-, the comparative-, the discernability- and the uncertainty analysis. There is strong evidence of the superiority of the pumps based on the modern technology. Recommendations and Outlook  The main source of the environmental impact in the case of pumps is the usage stage and the consumption of energy. That is why it should be the main area to improve. The LCA results show that actions taken in the usage stage and energy consumption can lead to a considerable reduction of the environmental impacts.     

HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions

Pseudomonas syringae pv. tomato DC3000 causes bacterial speck disease in tomato, and it elicits the hypersensitive response (HR) in non-host plants such as Nicotiana tabacum and Nicotiana benthamiana. The compatible and incompatible interactions of DC3000 with tomato and Nicotiana spp., respectively, result in plant cell death, but the HR cell death occurs more rapidly and is associated with effective plant defense. Both interactions require the Hrp (HR and pathogenicity) type III secretion system (TTSS), which injects Hop (Hrp outer protein) effectors into plant cells. Here, we demonstrate that HopPtoN is translocated into tomato cells via the Hrp TTSS. A hopPtoN mutant produced eightfold more necrotic 'speck' lesions on tomato leaves than did DC3000, but the mutant and the wild-type strain grew to the same level in infected leaves. In non-host N. tabacum leaves, the hopPtoN mutant produced more cell death, whereas a DC3000 strain overexpressing HopPtoN produced less cell death and associated electrolyte leakage in comparison with wild-type DC3000. Transient expression of HopPtoN via infection with a PVX viral vector enabled tomato and N. benthamiana plants to tolerate, with reduced disease lesions, challenge infections with DC3000 and P. syringae pv. tabaci 11528, respectively. HopPtoN showed cysteine protease activity in vitro, and hopPtoN mutants altered in the predicted cysteine protease catalytic triad (C172S, H283A and D299A) lost HR suppression activity. These observations reveal that HopPtoN is a TTSS effector that can suppress plant cell death events in both compatible and incompatible interactions.