Does increased endogenous CCK interact with serotonin to reduce food intake in rats?

The present study was aimed to test the hypothesis that increased endogenous CCK may interact with the anorectic serotonergic agent dl-fenfluramine to reduce food intake in rats. Previous studies, using selective CCK receptor antagonists, could demonstrate CCK-dependent 5-HT-induced anorexia. In the present approach, we used protease inhibitors to increase levels of endogenous CCK instead of blocking CCK receptors by antagonists. The protease inhibitors we used were soybean trypsin inhibitor (STI) and camostate. We hypothesized that combining the anorectic serotonergic drug dl-fenfluramine with either STI or camostate should result in an enhanced hypophagic effect when compared to single drug treatment. All feeding experiments were performed in non-deprived rats during night time feeding. Given alone, STI (500 mg/kg, po), camostate (200 mg/kg po) and also fenfluramine (1–9 mg/kg ip) reduced significantly food intake, with a more pronounced effect following fenfluramine. However, the experiments do not provide evidence for any additive or synergistic action between camostate or STI and the anorectic serotonergic drug dl-fenfluramine on food intake.     

Sensitive detection of C-reactive protein in serum by immunoprecipitation–microchip capillary gel electrophoresis

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP–MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R² = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP–MCGE bears potential for point-of-care diagnosis.     

Image-Guided Radiotherapy Using a Modified Industrial Micro-CT for Preclinical Applications

Purpose/ObjectiveAlthough radiotherapy is a key component of cancer treatment, its implementation into pre-clinical in vivo models with relatively small target volumes is frequently omitted either due to technical complexity or expected side effects hampering long-term observational studies. We here demonstrate how an affordable industrial micro-CT can be converted into a small animal IGRT device at very low costs. We also demonstrate the proof of principle for the case of partial brain irradiation of mice carrying orthotopic glioblastoma implants.Methods/MaterialsA commercially available micro-CT originally designed for non-destructive material analysis was used. It consists of a CNC manipulator, a transmission X-ray tube (10–160 kV) and a flat-panel detector, which was used together with custom-made steel collimators (1–5 mm aperture size). For radiation field characterization, an ionization chamber, water-equivalent slab phantoms and radiochromic films were used. A treatment planning tool was implemented using a C++ application. For proof of principle, NOD/SCID/γc^−/− mice were orthotopically implanted with U87MG high-grade glioma cells and irradiated using the novel setup.ResultsThe overall symmetry of the radiation field at 150 kV was 1.04±0.02%. The flatness was 4.99±0.63% and the penumbra widths were between 0.14 mm and 0.51 mm. The full width at half maximum (FWHM) ranged from 1.97 to 9.99 mm depending on the collimator aperture size. The dose depth curve along the central axis followed a typical shape of keV photons. Dose rates measured were 10.7 mGy/s in 1 mm and 7.6 mGy/s in 5 mm depth (5 mm collimator aperture size). Treatment of mice with a single dose of 10 Gy was tolerated well and resulted in central tumor necrosis consistent with therapeutic efficacy.ConclusionA conventional industrial micro-CT can be easily modified to allow effective small animal IGRT even of critical target volumes such as the brain.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis

Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.     

Differences in the Large Extracellular Loop between the K+-Cl? Cotransporters KCC2 and KCC4

K⁺Cl⁻ cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini. Here, we investigated sequence requirements of the large extracellular loop (LEL) for function in KCC2 and KCC4. Mutation of all four evolutionarily conserved cysteines abolished KCC2 transport activity. This behavior differs from that of its closest relative, KCC4, which is insensitive to this mutation. Chimeras supported the differences in the LEL of the two cotransporters, because swapping wild-type LEL resulted in functional KCC2 but rendered KCC4 inactive. Insertion of the quadruple cysteine substitution mutant of the KCC4 loop, which was functional in the parental isoform, abolished transport activity in KCC2. Dose-response curves of wild-type and chimeric KCCs revealed that the LEL contributes to the different sensitivity to loop diuretics; a KCC2 chimera containing the KCC4 LEL displayed an IC₅₀ of 396.5 μm for furosemide, which was closer to KCC4 (548.8 μm) than to KCC2 (184.4 μm). Cell surface labeling and immunocytochemistry indicated that mutations do not affect trafficking to the plasma membrane. Taken together, our results show a dramatic and unexpected difference in the sequence requirements of the LEL between the closely related KCC2 and KCC4. Furthermore, they demonstrate that evolutionarily highly conserved amino acids can have different functions within KCC members.