Experimental and clinical possibilities of MR spectroscopy of the heart

MR-spectroscopy of the heart is a relatively new technique for the study of various aspects of cardiac metabolism. The majority of results has so far been obtained with the isolated perfused heart. Here, 31P-MR spectroscopy can be employed to measure high-energy phosphate metabolism and intracellular pH repeatedly and non-invasively. Using a technique called saturation transfer, velocities of enzymatic reactions, such as the creatine kinase reaction, can be measured. Intra- and extracellular Na+ and K+ concentrations can be registered with 23Na- and 39K-MR in conjunction with shift reagent. 13C-MR can be used to tackle carbohydrate metabolism. In-situ-R-spectroscopy allows determination of high-energy phosphates in intact large mammals. Clinical applications of MR-spectroscopy remain to be defined; preliminary results indicate high diagnostic and prognostic potential for patients with coronary artery disease and congestive heart failure.     

Changes in the mental representations of relational behavior in depressive patients

Abstract

In this study the mental representations of interpersonal relations in depressive patients were assessed using the axis relationships of the operationalized psychodynamic diagnostics (OPD) system. The data were obtained at the beginning and at the end of inpatient psychotherapy. Although a link was assumed to exist between the severity of the symptoms and the variability of the mental representation of relational behavior, this was not proven. Also, contrary to expectations, an increase in the variability of the mental representations of the patients’ own relational behavior was not observed on completion of therapy. However, patients’ mental representation of their social partners’ relational behavior tends to become more variable. A shift in the focus of relationships was also observed.     

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.Neurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson’s disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1–4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein–coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin– and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G protein–independent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracellular loop 3 by a fusion protein, as the intracellular domain is otherwise too small to provide crystal contacts. The fusion protein approach has provided a wealth of valuable structural data on GPCRs, but as it renders the crystallized constructs signaling-inactive, the most important functionality—the activation of G proteins—cannot be confirmed for these structures. This leads inevitably to a degree of uncertainty regarding the physiological relevance of intracellular structural aspects, and it also impedes the elucidation of signaling mechanisms, as functional assays and structure determination cannot be performed with the same GPCR constructs.Crystallization in the absence of fusion proteins was so far mainly possible for rhodopsin (12), the A_2A adenosine receptor (A2AR) (13), and the β₁-adrenergic receptor (14). Together, they share a high stability, which is either given naturally (rhodopsin) or it is due to stabilizing mutations. High stability appeared to be crucial for crystallographic success, as it allowed the application of harsh short-chain detergents. These tend to form small micelles, which may explain why crystal contact formation can occur under these conditions despite the small extra- and intracellular domains of class A GPCRs.Besides the stability requirement and/or the necessity of fusion proteins, structural studies of GPCRs have also been complicated by the need of eukaryotic expression systems [e.g., Spodoptera frugiperda (Sf9) insect cells], as prokaryotes exhibit generally low functional expression levels of wild-type GPCRs. However, prokaryotes such as Escherichia coli offer several advantages compared with insect cells, including quick genetic modification strategies, growth to high cell densities, fast doubling times, inexpensive media, absence of glycosylation, and robust handling. Furthermore, E. coli is well suited for producing fully isotope-labeled proteins—a crucial requirement for many NMR studies, which are limited to date.To exploit these advantages, we recently developed a directed evolution method for high functional GPCR expression levels in E. coli (15). In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10⁸ different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16, 17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli. Selection pressure for high functional expression levels was applied by incubating the induced cells with fluorescently labeled neurotensin, which allowed enrichment of the best expressing cells by fluorescence-activated cell sorting (FACS). The outlined procedure was performed in cycles, leading to a gradual adaptation of the NTR1 population toward high functional expression levels, and additionally, it gave rise to an increase in thermostability for certain variants.In a second technology, termed CHESS (cellular high-throughput encapsulation, solubilization and screening), we adapted this concept to directly evolve NTR1 variants for high thermostability in short-chain detergent micelles—a property that is not only beneficial for structural studies but also for in vitro drug screening (18). The crucial development of CHESS was to surround, simultaneously, every E. coli cell by a semipermeable polysaccharide capsule. This allows us to solubilize the receptor mutants with harsh short-chain detergents, each mutant inside its own encapsulated cell, all at once and in the same test tube. Both the solubilized receptors and their encoding plasmids are maintained within the same capsules. Long-term incubation under these conditions followed by labeling of the encapsulated solubilized receptors with fluorescent neurotensin and rounds of FACS enrichment ensured a strong selection pressure and a gradual adaption of the NTR1 population toward high stability in harsh short-chain detergents (18).In this work, we present the crystal structures of three evolved NTR1 variants, which were either obtained by evolving high functional expression levels in E. coli or by directed evolution for stability in detergent micelles. In contrast to the majority of crystallized GPCRs, our NTR1 variants are devoid of bulky modifications at the cytoplasmic face and can thus remain signaling-active, which allows us to gain unique insights into the structure–function relationship of NTR1.     

Patterns of liver iron accumulation in patients with sickle cell disease and thalassemia with iron overload

The rate and pattern of iron deposition and accumulation are important determinants of liver damage in chronically transfused patients. To investigate iron distribution patterns at various tissue iron concentrations, effects of chelation on hepatic iron compartmentalization, and differences between patients with sickle cell disease (SCD) and thalassemia major (TM), we prospectively investigated hepatic histologic and biochemical findings in 44 patients with iron overload (35 SCD and 9 TM). The median hepatic iron content (HIC) in patients with TM and SCD was similar at 12.9 and 10.3 mg Fe/g dry weight, respectively (P = 0.73), but patients with SCD had significantly less hepatic fibrosis and inflammation (P < 0.05), less hepatic injury, and significantly less blood exposure. Patients with SCD had predominantly sinusoidal iron deposition, but hepatocyte iron deposition was observed even at low HIC. Chelated patients had more hepatocyte and portal tract iron than non‐chelated ones, but similar sinusoidal iron deposition. These data suggest that iron deposition in patients with SCD generally follows the traditional pattern of transfusional iron overload; however, parenchymal hepatocyte deposition also occurs early and chelation removes iron preferentially from the reticuloendothelium. Pathophysiological and genetic differences affecting iron deposition and accumulation in SCD and TM warrants further investigation ( http://www.clinicaltrials.gov # NCT00675038).     

58. Fibroadenoma intracanaliculare phyllodes mammae

Zusammenfassung Bei dem Fibroadenoma intracanaliculare phyllodes mammae (Cystosarcoma mammae) handelt es sich um eine verhältnismäßig seltene (MayoKlinik 13 Beobachtungen innerhalb von 40 Jahren) fibroepitheliale Geschwulst meist der weiblichen Brustdrüse. Die Besonderheit dieser Geschwulstform liegt darin, daß es hinsichtlich der Dignität völlig voneinander abweichende Verlaufsformen gibt. Auch histologisch ist die Unterscheidung zwischen der benignen und malignen Variante schwierig. Daher ist eine enge Zusammenarbeit mit dem Pathologen und dem Radiologen erforderlich. Eine eigene Beobachtung erstreckt sich auf eine 51 jährige Frau mit 2 jähriger Anamnese. Das Operationspräparat wog 3000 g. Die histologische Untersuchung sprach mehr für die gutartige Verlaufsform. 10 Monate nach der Operation war die Patientin noch beschwerde- und rezidivfrei.

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Cystosarcoma phyllodes mammae

Summary Fibroadenoma intracanaliculare phyllodes mammae (cystosarcoma of the breast) is a relatively rare (13 observations in the Mayo clinic within 40 years) fibroepithelial tumor, mostly of the female breast. The peculiarity of these tumours is their widely divergent natural histories. Histological differentiation between benign and malignant variants is difficult. For this reason close co-operation with the pathologist and the radiologist is necessary. I had observed a woman, 51 years of age, with a history of two years. The extirpated preparation weighed 3000 grams. Histological examination indicated a benign type. The patient was still free from symptoms and recurrences ten months after operation.