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Due to an increasing competence orientation of medical studies, surgical curricula are being adapted in many places. In addition to surgical knowledge and practical skills, these should also teach competencies in differential diagnostics and treatment. The teaching of surgical knowledge through lectures and seminars and the demonstration of practical skills, e.g., through the use of logbooks in the Bock Practical Surgery (BP), only allows limited active engagement with surgical competencies on differential diagnostics and treatment. A reflection-based portfolio allows, through the independent written elaboration of surgical topics, an active engagement with the competencies and promises a higher learning effect. In the context of the implementation of such a portfolio as part of the proof of activity in BP, the effects on the acquisition of competencies and on the way of learning were investigated.Using a mixed methods approach, we compared competence acquisition using a reflection-based portfolio with learning using a logbook. Students conducted a self-assessment of competencies using questionnaire surveys before and after the BP. Through focus group interviews with discussions among students using a guideline, we explored the different ways of acquiring competencies. In addition, the examination and evaluation results of both cohorts were compared.Students' self-assessed competency acquisition and examination and evaluation results showed no differences when comparing the two cohorts. During the focus group interviews, we were able to show that in the perception of the students, surgical competencies can be made more visible and thus more explicit with the help of a reflection-based portfolio. In addition, self-regulated learning was promoted without neglecting practical skills. Students demanded greater supervision and guidance by mentors in both groups.HINTERGRUND: Durch eine zunehmende Kompetenzorientierung des Medizinstudiums und der Weiterbildung werden chirurgische Currikula vielerorts angepasst. Diese sollten, neben chirurgischem Wissen und praktischen Fertigkeiten, auch Kompetenzen zu Differenzialdiagnostik und -therapie vermitteln. Die Vermittlung chirurgischen Wissens durch Vorlesungen und Seminare und der Nachweis praktischer Fertigkeiten, z. B. mithilfe des Einsatzes von Logbüchern im Blockpraktikum (BP) Chirurgie, führt nur eingeschränkt zu einer aktiven Auseinandersetzung mit den chirurgischen Kompetenzen zu Differenzialdiagnostik und -therapie. Ein reflexionsbasiertes Portfolio kann, durch die eigenständige schriftliche Bearbeitung chirurgischer Themen eine aktive Auseinandersetzung mit den Kompetenzen ermöglichen und verspricht einen höheren Lerneffekt. Bei der Implementierung eines solchen Portfolios im Rahmen des Tätigkeitsnachweises im BP wurden die Effekte auf den Kompetenzerwerb und auf die Art und Weise des Lernens untersucht.Mit einer Kombination aus einer quantitativen und einer qualitativen Methode („mixed methods“) haben wir den Einsatz eines Logbuchs im BP Chirurgie mit dem Einsatz eines reflexionsbasierten Portfolios verglichen. Mittels Fragebogenerhebungen vor und nach dem BP erfolgte durch die Studierenden eine Selbsteinschätzung von Kompetenzen. Im Rahmen von Fokusgruppeninterviews anhand eines Leitfadens, mit Diskussionen unter Studierenden, haben wir die unterschiedlichen Wege des Kompetenzerwerbs untersucht. Zusätzlich wurden die Prüfungs- und Evaluationsergebnisse beider Kohorten verglichen.Der in der Selbsteinschätzung erhobene Kompetenzerwerb der Studierenden und die Prüfungs- und Evaluationsergebnisse zeigten im Vergleich beider Kohorten keinen Unterschied. Im Rahmen der Fokusgruppeninterviews konnten wir zeigen, dass in der Wahrnehmung der Studierenden chirurgische Kompetenzen mithilfe eines reflexionsbasierten Portfolio sichtbarer und damit eindeutiger gemacht werden können. Zusätzlich wurde selbstreguliertes Lernen der Studierenden gefördert, ohne dass praktische Fertigkeiten vernachlässigt wurden. Eine stärkere Supervision und Anleitung durch Mentor:innen wurde in beiden Gruppen gefordert.

Bei der Osteosynthese von Tibiakopffrakturen mit Gelenkbeteiligung konnen sich nicht-rekonstruierbare osteochondrale Defekte zeigen. Dabei erfolgt die Behandlung ossarer Defekte als Goldstandard mittels autologer Spongiosaplastik. Eine gezielte primare knorpelregenerative Therapie bei der osteosynthetischen Versorgung von Tibiakopffrakturen ist in der Literatur bisher nicht beschrieben. In diesem Beitrag wird der Fall eines 36-jahrigen mannlichen Patienten vorgestellt, der sich beim Wakeboarden eine komplexe bikondylare Tibiakopffraktur zugezogen hatte. Es wurde eine arthroskopisch assistierte Osteosynthese durchgefuhrt. Einzeitig wurde ein nichtrekonstruierbarer osteochondraler Defekt primar mittels autologer Spongiosa aufgefullt und anschliesend mittels einer zellfreien Kollagenmatrix im AMIC®-Verfahren (autologe matrixinduzierte Chondrogenese) abgedeckt. Der postoperative Verlauf war komplikationslos. Nach 10 Monaten konnte uneingeschrankte Sportfahigkeit wiedererlangt werden. Eine Magnetresonanztomographie (MRT) und eine Arthroskopie im Rahmen der Materialentfernung zeigten eine stufenfreie Gelenkflachenrekonstruktion sowie ein suffizientes Knorpelregenerat des lateralen Tibiaplateaus. Der vorliegende Fallbericht zeigt, dass traumatische osteochondrale Defekte mittels autologer Spongiosaplastik in Kombination mit einer AMIC® zum Zeitpunkt der Osteosynthese zur Ausheilung gebracht werden konnen.

We present the case of an adolescent male developing an ankylosis of the knee after septic arthritis following anterior cruciate ligament reconstruction (ACLR). The patient was shifted to our institution with postoperative septic arthritis associated with a systemic septic condition. Before, repeated arthroscopic surgery had been conducted without any improvement. MRI showed a concomitant osteomyelitis. The infection (Gachter IV, Staphylococcus aureus) was controlled by an open surgical approach and graft removal. An increasing joint stiffness was documented. X-rays showed an ankylosis at 30° of flexion and early closure of growth plates. Functional knee scores showed significantly worse results. Early diagnosis and a stage-adapted treatment in septic arthritis following ACLR are mandatory. In advanced stages or concomitant osteomyelitis an open approach and graft removal may be appropriate. The antibiotic treatment should be adapted consistently.

Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic–pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 μg) resulted in an 18-fold stimulation (P < 0.05) of progesterone (P4) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 (n = 10) or saline (n = 8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable (P < 0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation (P < 0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P4 concentrations were not different (P = 0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.

We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release.

Concept and design of an independent scientific evaluation of different pathways of care for schizophrenia patients in Germany with respect to effectiveness and efficiency are presented. In this prospective, observational study, schizophrenia patients receiving an integrated care treatment, the intervention group (IG), are compared with patients under routine care conditions treated by the same physician (first control group, CG 1). A second control group (CG 2) of patients treated by office-based psychiatrists not participating in the integrated care program will be recruited and their data compared with the two other groups. The total amount of psychiatric hospital days after 12 months is defined as primary outcome parameter. Secondary outcome parameters comprise the frequency of psychiatric inpatient readmissions, severity of schizophrenia symptoms, remission rates and quality of life. Patients undergo assessments at baseline, month 6 and 12 using standardized and experimental questionnaires. Routine data of a regional German social health insurance fund complement information on included patients. Additionally, a cost-effectiveness and cost-utility analysis will be performed. Until now, 137 psychiatrists included 980 patients in the integrated care project in Lower Saxony, Germany, and 47 psychiatrists (IG and both CGs) are willing to participate in the independent evaluation. For the first time, a prospective observational controlled evaluation study of a countrywide integrated care project planning to recruit 500 schizophrenia patients has started using comprehensive assessments as well as routine data of a social health insurance fund.

Most peptide and protein drugs are short-lived species in vivo with a circulatory half-life of several minutes. This is particularly valid for non-glycosylated proteins with a molecular mass of less than 50 kDa. Since peptide/protein drugs are not absorbed orally, prolonged maintenance of therapeutically active drugs in the circulatory system is of primary clinical importance. Another major obstacle of injected polypeptide drugs is the elevated concentration of 100–1000 times above the therapeutical level that may be present in the circulatory system shortly after administration. Such overdosing may lead to undesirable side effects such as over-stimulation or down-regulation of receptor sites. In this review we describe two new strategies that overcome these two problems of systemically injected peptide/protein drugs. The first strategy includes Fmoc and FMS derivatization of peptides, proteins and low molecular-weight drugs, converting them to inactive prodrugs that undergo reactivation with desirable pharmacokinetic patterns in body fluids. Based on this Fmoc/FMS-technology, we have developed a second strategy, reversible pegylation. Inactive pegylated peptide/protein drugs release the native active parental molecule at slow rates, and in homogeneous fashion under physiological conditions, thus facilitating prolonged therapeutic effects, following a single administration.

We present the case of an adolescent male developing an ankylosis of the knee after septic arthritis following anterior cruciate ligament reconstruction (ACLR). The patient was shifted to our institution with postoperative septic arthritis associated with a systemic septic condition. Before, repeated arthroscopic surgery had been conducted without any improvement. MRI showed a concomitant osteomyelitis. The infection (Gachter IV, S. aureus) was controlled by an open surgical approach and graft removal. An increasing joint stiffness was documented. X-rays showed an ankylosis at 30° of flexion and early closure of growth plates. Functional knee scores showed significantly worse results. Early diagnosis and a stage-adapted treatment in septic arthritis following ACLR are mandatory. In advanced stages or concomitant osteomyelitis, an open approach and graft removal may be appropriate. The antibiotic treatment should be adapted consistently.

Biomimetic analogs 1 of the microbial siderophore (iron carrier) ferrichrome were labeled with a fluorescent marker at a site which does not interfere with iron binding or receptor recognition to provide iron(III) carriers 5 and 10 (Figure 1). These carriers were built from a tetrahedral carbon as an anchor which was symmetrically extended by three converging iron-binding chains and a single, exogenous anthracenyl residue. Carriers 5 varied in the nature of the amino acids (G = glycyl, A = alanyl and L = leucyl) linking the anchor with the iron-binding hydroxamate groups, while the alanyl derivative 10A differed from 5A in the spacer between the anthracenyl label and the anchor. Examination of these binders by 1H NMR confirmed that their conformations were analogous to those of the nonlabeled parent compounds. Titration experiments using UV/vis and fluorescence spectroscopy demonstrated the quenching of these compounds' fluorescence upon iron(III) loading and its recovery upon iron(III)'s release to a com...

Molecular recognition and signaling are at the heart of biological processes as they govern intercellular communication and information transfer. Here we probe the recognition phenomena of siderophore-mediated microbial iron(1II) uptake, which involves two recognition processes: recognition of iron(II1) by microbial siderophores (iron(II1) carriers), and recognition and binding of siderophomiron(II1) complexes by microbial membrane receptors. In addition, we introduce fluorescent siderophore analogs that respond to iron binding with changes of their fluorescence and thereby provide artificial binders that combine iron nxogni tion with signaling.

Testicular cancer is one of the commonest cancers in men of working age, and is increasing in incidence in Europe and North America. One suggested mechanism of causation is that there is impaired differentiation of germ cells in the pre- or perinatal period, followed by malignant transformation in later life, possibly by a hormonal mechanism. Endocrine disrupting chemicals (EDCs) have been a major focus of interest for etiological research into testicular cancer because they interact with various hormonal pathways. Several EDCs including bisphenol A, phthalates, metals, polychlorinated biphenyls, and organochlorines have been investigated, but there are few studies and those that exist have not been able to assess exposure well. In addition, several studies, particularly those with better exposure assessment, have suggested that workers in electrical occupations have increased risks of testicular cancer. Electromagnetic radiation may have subthermal effects or may disrupt hormone release. Chronodisruption such as due to shift-work could potentially increase the risk of testicular cancer via disruption of hormonal cycles, but only one study has so far investigated this possibility. Lastly, solvent exposure, particularly to dimethylformamide, has been suggested to be associated with testicular cancer, but almost all these studies are based on job title only, with no specific assessment of solvent exposure. In conclusion, there is little evidence available on which to base definitive statements about occupational causes of testicular cancer. Future studies need to improve exposure assessment and develop ways to adjust for possible prenatal factors.

Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.

We assessed in vivo antimalarial action of a lipophilic iron (III) chelator belonging to a new synthetic family of biomimetic siderophores previously termed reversed siderophores (RSFs). The family member, RSF ileum2, was chosen for its high membrane permeability and fast irreversible inhibition of human malaria parasite growth in vitro. [Shanzer A, et al., Proc Natl Acad Sci USA 88:6585, 1991 and Lytton SD, et al., Blood 81:214, 1993]. The lipophilic drug was administered to Swiss mice by subcutaneous route in fractionated coconut oil at a dosage of 0.37 g/kg every 8 hr with no adverse reactions observed. After 3-4 injections demonstrable suppression of Plasmodium vinckei petteri infection was observed and an additional 3-4 injections resulted in 2-3-fold lower parasitemia with prolonged survival time over sham-injected control mice.

1-{[(5-Nitro-2-furanyl)methylene]amino}-1,2,4-triazole, C7H5N5O3, is a new analogue of nifurtimox with activity against Tripanosoma cruzi. In the crystal structure, the molecule lies on a mirror plane and has an E–sZ conformation along the N=C—C moeity. The molecules are linked through C—H⋯O and C—H⋯N interactions.

We present here the physicochemical and biochemical properties of NBD-DFO, the 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of the siderophore, desferrioxamine B (DFO) (Lytton et al., Mol. Pharmacol. 40, 584, 1991). Modification of DFO at its terminal amine renders it more lipophilic, imparts to it fluorescent properties, and is conservative of the high-affinity iron(III) binding capacity. NBD-DFO partitions readily from aqueous solution into n-octanol (Pcoeff = 5) and displays solvent-induced shifts in absorption and fluorescence spectra. The relative quantum yield of the probe's fluorescence increases over a 10-fold range with decreasing dielectric constant of the solvent. Fluorescence is quenched upon binding of iron(III) to the probe. We demonstrate here the application of NBD-DFO for the specific detection and monitoring of iron (III) in solutions and iron(III) mobilization from cells. Interactions between fluorescent siderophore and the ferriproteins ferritin and transferrin were monitored under physiological conditions. Iron removal from ferritin was evident by the demonstrable quenching of NBD-DFO fluorescence by scavenged iron(III). Quantitation of iron sequestered from cells by NBD-DFO or from other siderophore-iron(III) complexes was accomplished by dissociation of NBD-DFO-Fe complex by acidification and addition of excess ethylenediamin-etetraacetic acid. The sensitivity of the method and the iron specificity indicate its potential for monitoring chelatable iron under conditions of iron-mediated cell damage, iron overload, and diseases of iron imbalance such as malaria.

The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a beta-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3(IIIB) peptide bound to the 0.5beta antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3(MN) and a V3(CONSENSUS) peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3(CONSENSUS) epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks the GPGR segment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.

The aim of occupational epidemiology is to describe workplace-related diseases and to identify their underlying causes. Its primary goal is to protect workers from hazardous effects of the working process by applying work-related primary and secondary prevention measures. To assess health risks different study designs and a wide array of complex study instruments and methods are frequently employed that cannot be replaced by toxicological investigations. This paper primarily addresses health risks by agent exposures. In this context a central task of occupational epidemiology is careful assessment of exposure. Different data sources, such as work site measurements, register data, archive material, experts' opinion, and the workers' personal estimates of exposure may be used during this process. In addition, biological markers can complement exposure assessment. Since thorough occupational epidemiologic studies allow assessment of disease risks under realistic exposure conditions, their results should be more frequently used to derive workplace-related threshold limit values.

Quantitative structure-activity relationships were performed on a set of 5-nitrofuran derivatives. The correlation parameters used were hydrophobicity and connectivity. The former was measured by means of the capacity factor (log ko′) obtained by HPLC and the latter through the molecular valence connectivity index1χv obtained by computational methods. The biological activity data considered were growth inhibition percentages ofT. cruzi (GI % (48 h)) and half-maximal inhibitory concentration of Glutathione Reductase (I0.5 (max)). Good adjustment, r=0.98, was obtained if a logarithmic model is used for I0.5 (max) correlations. When GI% is considered, correlations were poorer and the consideration of both structural parameters was necessary for their improvement, r=0.94.

Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5beta, raised against gp120 of the X4 isolate HIV-1(IIIB), neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3(MN) and V3(IIIB), adopt alternative beta-hairpin conformations when bound to 447-52D and 0.5beta, respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3(IIIB) peptide bound to 447-52D was determined by NMR. V3(IIIB) and V3(MN) peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3(IIIB) peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5beta. Comparison of the three known V3 structures leads to a model in which a 180 degrees change in the orientation of the side chains and the resulting one-residue shift in hydrogen bonding patterns in the N-terminal strand of the beta-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, I314, and I316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5beta with variable residues underlie its strain specificity.

Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.

All organisms need to acquire iron in a bioavailable form and integrate it into biologically active molecules1. Microorganisms and plants have developed elaborate mechanisms for the synthesis, secretion and uptake of siderophores whose task is to sequester extracellular iron and mobilize it into cells as iron-complexes by receptor and translocation-mediated processes2. Mammalian cells have devised analogously regulated paths for obtaining iron from body fluids by receptor-mediated endocytosis of circulating iron-transferrin3 and possibly also by other membrane associated transport mechanisms4,5. The fate of the acquired iron depends on the metabolic status of cells and the route of entry and delivery of the metal into cell compartments. In higher organisms, regulation of cytosolic iron comprises primarily complementary mechanisms for the translational control of synthesis of proteins involved in iron intake and those involved in cellular iron storage6.

We have designed two subfamilies of lipophilic iron (III) chelators previously termed reversed siderophores (RSFs). The agents display physicochemical properties that favor extraction of iron beyond membrane barriers of Plasmodium falciparum-infected red blood cells. We studied the in vitro antimalarial potency of RSFs and their relationship to the membrane permeation properties of these agents. The mode of RSF action involves: (1) fast access to intracellular compartments of parasitized cells; (2) selective and high-affinity chelation of iron (III) from parasitized cells; (3) fast exit from cells after iron (III) complexation; and (4) exertion of cell damage on parasites exposed for 3 to 5 hours to drugs, irrespective of the stage of parasite development. These results suggest that on reaching a critical intraerythrocyte target, RSFs induce an iron deficit that parasites in general, and rings in particular, have limited capacity to restore.

Electrochemical studies (Formal Potentials and Coulometry) were performed on several analogues of Nifurtimox, in order to gain insight on the proposed mechanism of action of these drugs. It is thought that Nifurtimox shows part of its trypanocidal activity via an electron transfer process, leading to the production of active oxygen species that kill the parasite. Formal potentials provide relevant data about the feasibility of electron transfer in vivo. All analogues tested contained the nitro group, which is the most easily reducible group in the parent drug Nifurtimox. The formal potentials values determined ranged from -1.86 to -0.46 V. Coulometric runs were carried out to study the one-electron transfer process. A good correlation between formal potentials and biological activity was found.

The number and size of follicles selected for ovulation differ between species. The aim of this study was to characterise antral follicular growth and determine the size when selection of the ovulatory follicle occurs in the monovular brushtail possum. For this study, antral follicles ≥ 1 mm were dissected from the ovaries of 31 adult female possums at different reproductive states and follicular fluid and granulosa cells were harvested from each individual follicle. Selection of the ovulatory follicle in the brushtail possum occurred when follicles reached between 2.5 and 2.8 mm in diameter. Based on the analysis of steroids in follicular fluid, before selection, most follicles produced varying amounts of oestradiol (E2), but very few if any produced progesterone (P4). After selection, the selected follicle continued to produce increasing amounts of E2 and P4, whereas most other follicles were steroidogenically inactive. Near the time of ovulation, presumably after the LH surge, P4 became the predominant steroid produced by the selected follicle and most other follicles once again produced varying amounts of E2. The number of granulosa cells per follicle was highly variable, but tended to increase with increasing diameter. Cell viability was very high, averaging about 95%. Interestingly, the morphology of granulosa cells changed markedly after selection becoming larger and granular in appearance. The weights of the vaginal cul-de-sac and uteri correlated well with the presence of a selected follicle. In ovaries from pregnant animals (n = 3), follicles grew up to 3.5 mm, and although they reached the size of a selected follicle during the follicular phase, E2 production by the other follicles was not suppressed and the weights of the cul-de-sac were less than those from non-pregnant animals with similar sized follicles. During anoestrus (n = 4), follicles did not grow beyond 2 mm and produced very little steroids.

Ten new derivatives 1 structurally related to Nifurtimox have been synthesized from 5-nitrofurfural and the corresponding N-aminoheterocyclic compounds. Physical data, spectroscopic characteristics and biological properties have been examined. An unusual long-range coupling constant in the pyrazolyl derivative 1b has been observed. Compounds in which the heterocyclic counterparts are 1,2,4-triazol-4-yl and pyridin-1-yl groups clearly show s superior activity against Trypanosoma cruzi. Neue Nifurtimox-Analoge mit Wirkung gegen Trypanosoma cruzi Zehn neue Derivate strukturbezogen auf Nifurtimox wurden aus 5-Nitrofurfural und den entspr. heterocyclischen N-amino Verbindungen hergestellt. Uber ihre physikalischen und spektroskopischen Daten bzw. biologische Wirkung wird berichtet. Eine ungewohnliche “long-range” Kopplungs Konstante wurde an der Pyrazolylverbindung 1b beobachtet. Die Verbindungen, in denen der heterocyclischer Partner 1,2,4-Triazol-4-yl und Pyridin-1-yl Gruppierungen sind, zeigen eine eindeutig hohere Wirkung gegenuber Trypanosoma cruzi als Nifurtimox.

Ten new derivatives 1 structurally related to Nifurtimox have been synthesized from 5-nitrofurfural and the corresponding N-aminoheterocyclic compounds. Physical data, spectroscopic characteristics and biological properties have been examined. An unusual long-range coupling constant in the pyrazolyl derivative 1b has been observed. Compounds in which the heterocyclic counterparts are 1,2,4-triazol-4-yl and pyridin-1-yl groups clearly show s superior activity against Trypanosoma cruzi. Neue Nifurtimox-Analoge mit Wirkung gegen Trypanosoma cruzi Zehn neue Derivate strukturbezogen auf Nifurtimox wurden aus 5-Nitrofurfural und den entspr. heterocyclischen N-amino Verbindungen hergestellt. Uber ihre physikalischen und spektroskopischen Daten bzw. biologische Wirkung wird berichtet. Eine ungewohnliche “long-range” Kopplungs Konstante wurde an der Pyrazolylverbindung 1b beobachtet. Die Verbindungen, in denen der heterocyclischer Partner 1,2,4-Triazol-4-yl und Pyridin-1-yl Gruppierungen sind, zeigen eine eindeutig hohere Wirkung gegenuber Trypanosoma cruzi als Nifurtimox.

1-¿[(5-Nitro-2-furyl)methylene]amino¿-1,2,4-triazole, C7H5N5O3, is a new analogue of nifurtimox with activity against Tripanosoma cruzi. In the crystal structure, the molecule lies on a mirror plane and has an E-sZ conformation along the N = C-C moiety. The molecules are linked through C-H...O and C-H...N interactions.

We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron (III) chelator with improved membrane permeation properties. Upon binding of iron (III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron (III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium falciparum were 3 +/- 1 microM for MA-DFO compared with 30 +/- 8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 microM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to approximately 10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DFO accomplishes faster than DFO, in accordance with its higher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium.