Deletion of the 6-kDa subunit affects the activity and yield of the bc1 complex from Rhodovulum sulfidophilum.

The cytochrome bc1 complex from Rhodovulum sulfidophilum purifies as a four-subunit complex: the cytochrome b, cytochrome c1 and Rieske iron-sulphur proteins, which are encoded together in the fbc operon, as well as a 6-kDa protein. The gene encoding the 6-kDa protein, named fbcS, has been identified. It is located within the sox operon, which encodes the subunits of sarcosine oxidase. The encoded 6-kDa protein is very hydrophobic and is predicted to form a single transmembrane helix. It shows no sequence homology to any known protein. The gene has been knocked-out of the genome and a three-subunit complex can be purified. This deletion leads to a large reduction in the yield of the isolated complex and in its activity compared to wild-type. The high quinone content found in the wild-type complex is, however, maintained after removal of the 6-kDa protein. Surprisingly, a fourth subunit of approximately 6 kDa is again found to copurify with the Rhv. sulfidophilum bc1 complex when only the fbc operon is expressed heterologously in a near-relative, Rhodobacter capsulatus, which lacks this small subunit in its own bc1 complex.     

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.     

High dopamine transporter selectivity can be displayed by remarkably simple non-nitrogen containing inhibitors

A series of 2-(3,4-dichlorophenyl)-cyclopent-1-enyl carboxylic acid esters and amides were prepared and tested for binding to the DAT, SERT, and NET. The achiral compounds were easily attained and found to inhibit DAT binding with K(i)-values ranging from 0.095 to 0.00003 mM. Among the compounds tested 2-(3,4-dichlorophenyl)-cyclopent-1-enyl carboxylic acid 2-methylphenyl ester was found to be highly selective with SERT/DAT>7000; NET/DAT>1700, K(i)=60 nM.     

Development and characterization of highly polymorphic long TC repeat microsatellite markers for genetic analysis of peanut

Background

The immune system has paradoxical roles during cancer development and the prognostic significance of immune modulating factors is controversial. The aim of this study was to determine the expression of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells.

Methods

On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding lymph node metastases. COX-2 was recorded as graded staining intensity.

Results

The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67 was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P < 0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with breast cancer mortality.

Conclusions

Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer.     

Insulin resistance in adolescents with Down syndrome: a cross-sectional study

Background

The prevalence of diabetes mellitus is higher in individuals with Down syndrome (DS) than in the general population; it may be due to the high prevalence of obesity presented by many of them. The aim of this study was to evaluate the insulin resistance (IR) using the HOMA (Homeostasis Model Assessment) method, in DS adolescents, describing it according to the sex, body mass index (BMI) and pubertal development.

Methods

15 adolescents with DS (8 males and 7 females) were studied, aged 10 to 18 years, without history of disease or use of medication that could change the suggested laboratory evaluation. On physical examination, the pubertal signs, acanthosis nigricans (AN), weight and height were evaluated. Fasting plasma glucose and insulin were analysed by the colorimetric method and RIA-kit LINCO, respectively. IR was calculated using the HOMA method. The patients were grouped into obese, overweight and normal, according to their BMI percentiles. The EPIINFO 2004 software was used to calculate the BMI, its percentile and Z score.

Results

Five patients were adults (Tanner V or presence of menarche), 9 pubertal (Tanner II – IV) and 1 prepubertal (Tanner I). No one had AN. Two were obese, 4 overweight and 9 normal. Considering the total number of patients, HOMA was 1.7 ± 1.0, insulin 9.3 ± 4.8 μU/ml and glucose 74.4 ± 14.8 mg/dl. The HOMA values were 2.0 ± 1.0 in females and 1.5 ± 1.0 in males. Considering the nutritional classification, the values of HOMA and insulin were: HOMA: 3.3 ± 0.6, 2.0 ± 1.1 and 1.3 ± 0.6, and insulin: 18.15 ± 1.6 μU/ml, 10.3 ± 3.5 μU/ml and 6.8 ± 2.8 μU/ml, in the obese, overweight and normal groups respectively. Considering puberty, the values of HOMA and insulin were: HOMA: 2.5 ± 1.3, 1.4 ± 0.6 and 0.8 ± 0.0, and insulin: 13.0 ± 5.8 μU/ml, 7.8 ± 2.9 μU/ml and 4.0 ± 0.0 μU/ml, in the adult, pubertal and prepubertal groups respectively.

Conclusion

The obese and overweight, female and adult patients showed the highest values of HOMA and insulin.     

The Escherichia coli SRP Receptor Forms a Homodimer at the Membrane

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Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp⁹⁸. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe³³⁵, and the pocket lined by Ala¹⁷³ and Thr⁴³⁹ is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).     

Vitamin B6 biosynthesis by the malaria parasite Plasmodium falciparum: biochemical and structural insights

Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a glutamine amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5 '-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 A. The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.     

The cytochrome bc1 complex from Rhodovulum sulfidophilum is a dimer with six quinones per monomer and an additional 6-kDa component.

A highly active, large-scale preparation of cytochrome bc1 complex has been obtained from the photosynthetic purple bacterium Rhodovulum (Rhv.) sulfidophilum. It has been characterized using mass spectrometry, quinone and lipid analysis as well as inhibitor binding. About 35 mg of pure complex can be obtained from 1 g of membrane protein. EPR spectroscopy and optical titrations have been used to obtain the redox midpoint potentials of the cofactors. The Em-value of 310 mV for the Rieske protein is the most positive midpoint potential for this protein in a bc1 complex so far. The bc1 complex from Rhv. sulfidophilum is very stable and consists of three subunits and a 6-kDa polypeptide. The complex appears as a dimer in solution and contains six quinone molecules per monomer which are tightly bound. EPR spectroscopy shows that the Q(o) site is highly occupied. High detergent concentrations convert the complex into an inactive, monomeric form that has lost the Rieske protein as well as the quinones and the 6-kDa component.