Increases in 1H-NMR mobile lipids are not always associated with overt apoptosis: evidence from MG-63 human osteosarcoma three-dimensional spheroids exposed to a low dose (2 Gy) of ionizing radiation

The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.     

Analysis of the periprosthetic femoral bone reaction after uncemented total hip arthroplasty with computertomography assisted osteodensitometry in vivo: 6-year follow-up]

AIMS: We prospectively analyzed the cancellous and cortical periprosthetic femoral bone reaction after implantation of a cementless total hip arthroplasty with computertomography assisted osteodensitometry after a mean of 1 and 6 years. MATERIALS AND METHODS: Twenty-one patients (? age at implantation: 52 years) with osteoarthrits of the hip joint received 21 cementless hip prostheses with a three-dimensionally tapered design. All patients were analyzed clinically, with CT-osteodensitometry and plain radiography after a mean of 10 days, at 1 and 6 years postoperatively. Cancellous and cortical bone density was evaluated automatically using a special software tool. RESULTS: The proximal region of the stem showed progessive cortical (? -15% 1 year, -25% 6 years post-OP) and cancellous (? -26% 1 year, -49% 6 years post-OP) bone density loss. Cortical bone density loss was lower and non-progressive at the diaphysis (? -7% 1 year, -9% 6 years post-OP) and the distal region (? -6% 1 year, -4% 6 years post-OP) of the stem. All stems showed no signs of loosening on plain radiography and good clinical results according to the Harris hip score. CONCLUSION: Computertomography assisted osteodensitometry is the only method which allows discrimination between periprosthetic cortical and cancellous bone density changes in vivo. The analyzed uncemented stem fixates at the diaphysis and distal region. Due to the changed biomechanical loading after stem implantation, progressive proximal cancellous bone density loss was measured for the first time in vivo. Its role in the pathogenesis of implant loosening is still unknown and needs to be further elucidated.     

Comparative Study of Alkaline, Saline, and Mixed Saline–Alkaline Stresses with Regard to Their Effects on Growth, Nutrient Accumulation, and Root Morphology of Lotus tenuis

Both saline and alkaline conditions frequently coexist in nature; however, little is known about the effects of alkaline and salt?Calkaline stresses on plants. We performed pot experiments with four treatments, control without salt addition and three stress conditions??neutral, alkaline, and mixed salt?Calkaline??to determine their effects on growth, nutrient accumulation and root architecture in the glycophytic species Lotus tenuis. Neutral and alkaline salts produced a similar detrimental effect on L. tenuis growth, whereas the effect of their combination was synergistic. Neutral salt addition, alone or mixed with NaHCO₃, led to significant leaf Na⁺ build up and reduced K⁺ concentration. In contrast, in plants treated with NaHCO₃ only, Na⁺ levels and the Na⁺/K⁺ ratio remained relatively unchanged. Proline accumulation was not affected by the high pH in the absence of NaCl, but it was raised by the neutral salt and mixed treatments. The total root length was reduced by the addition of NaCl alone, whereas it was not affected by alkalinity, regardless of the presence of NaCl. The topological trend showed that alkalinity alone or mixed with NaCl turned the root more herringbone compared with control roots, whereas no significant change in this index was observed in the treatment with the neutral salt only. The pattern of morphological changes in L. tenuis root architecture after the alkaline treatment (in the absence of NaCl) was similar to that found in the mixed salt?Calkaline treatment and different from that observed in neutral salt. A unique root morphological response to the mixed salt?Calkaline stress was the reduction in the ratio between xylem vessels and root cross-sectional areas.     

Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.     

Continued optimization of the MLPCN probe ML071 into highly potent agonists of the hM1 muscarinic acetylcholine receptor

This Letter describes the continued optimization of the MLPCN probe molecule ML071. After introducing numerous cyclic constraints and novel substitutions throughout the parent structure, we produced a number of more highly potent agonists of the M(1) mACh receptor. While many novel agonists demonstrated a promising ability to increase soluble APPα release, further characterization indicated they may be functioning as bitopic agonists. These results and the implications of a bitopic mode of action are presented.     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

Increasing healthy eating vs. reducing high energy-dense foods to treat pediatric obesity

Objective: The objective was to compare targeting increased eating of healthy foods vs. reducing intake of high energy‐dense foods within the context of a family‐based behavioral weight control program. Methods and Procedures: Forty‐one 8–12 year‐old children >85th BMI percentile were randomly assigned to a 24‐month family‐based behavioral treatment that targeted increasing fruits and vegetables and low‐fat dairy vs. reducing intake of high energy‐dense foods. Results: Children in the increase healthy food group showed greater reduction in zBMI compared to children in the reduce high energy‐dense food group at 12‐ (?0.30 zBMI units vs. ?0.15 zBMI units, P = 0.01) and 24‐ (?0.36 zBMI units vs ?0.13 zBMI units, P = 0.04) month follow‐up. Parents in the increase healthy food group showed greater reductions in concern about child weight (P = 0.007), and these changes were associated with child zBMI change (P = 0.008). Children in the reduce high energy‐dense group showed larger sustained reductions in high energy‐dense foods (P < 0.05). Baseline levels of high energy‐dense foods (P < 0.05), parent food restraint (P = 0.01), parent concern over parent weight (P = 0.01) and parent acceptance of the child (P < 0.05) moderated child zBMI change, with greater sustained reductions in zBMI for children in the increase healthy food group for each measure. Parent zBMI change followed the same pattern as child changes, and parent and child zBMI changes were correlated (P < 0.001). Discussion: Focusing on healthy food choices within an energy restricted diet may be useful in family‐based weight control programs.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.