1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Integration site of polyoma virus DNA in the inducible LPT line of polyoma-transformed rat cells

The structure of the polyoma virus (Py) integration site in the inducible LPT line of Py-transformed rat cells was determined by biochemical methods of gene mapping. LPT cell DNA was digested with various restriction enzymes. The digestion products were electrophoresed in agarose gels and transferred onto nitrocellulose sheets by Southern blotting. Fragments containing viral or cell DNA sequences, or both, were identified by hybridization with Py DNA or with a cloned flanking cell DNA probe. Cleavage of LPT DNA with enzymes that restrict the Py genome once generated linear Py DNA molecules and two fragments containing both cell and viral DNA sequences. Cleavage of LPT DNA with enzymes which do not restrict Py DNA generated series of fragments whose lengths were found to differ by increments of a whole Py genome; the smallest fragment in each series was found to be longer than the viral genome. These data indicate that LPT cultures contain Py insertions of various lengths integrated into the same chromosomal site in all the cells. The length heterogeneity of the viral insertions is due to the presence of 0, 1, 2, 3. . . Py genomes arranged in a direct tandem repeat within invariable sequences of viral DNA. Double-digestion experiments were also carried out with the above enzymes and with enzymes that cleave the Py genome at multiple sites. The data obtained in these experiments were used to construct a physical map of the integration site. This map showed that the early region of the virus remained intact even in the smallest insertion (which contains no whole duplicated genomes), whereas the late region was partially duplicated and split during integration. The smallest insertion is colinear with the Py physical map over a region including the entire Py genome and at least a part of the duplicated segment. This structure could give rise to nondefective circular viral DNA molecules by single homologous recombination events. Similar recombination events may occur at a higher frequency in the longer insertions, which include longer regions of homology, and may yield many more free viral genomes. The presence of these insertions in LPT cells could thus be one of the factors which account for the high inducibility of the LPT line.     

Protective effects of taxifolin on pazopanib-induced liver toxicity: an experimental rat model

Pazopanib is a tyrosine kinase inhibitor that is generally used for the treatment of metastatic renal cell cancer and advanced soft tissue sarcoma. It can cause various degrees of hepatotoxicity. Our study aimed to investigate the effect of taxifolin on pazopanib-induced liver toxicity. A total of 18 rats were divided into three groups: the pazopanib (PP), pazopanib plus taxifolin (TPP), and control (C) group. Taxifolin was administered to the TPP (n=6) group with a dose of 50 mg/kg. Distilled water was orally admnistered to the C (n=6) and PP (n=6) groups as a solvent. Subsequently, pazopanib 200 mg/kg was administered to the TPP and PP groups via the stomach. This procedure was repeated once a day for four weeks. Then, all rats were sacrificed, and their livers were removed. Malondialdehyde (MDA), total glutathione (tGSH), total oxidant status (TOS), and total antioxidant status (TAS) levels were evaluated. MDA and TOS levels were higher in the PP group compared with the levels of the other parameters (P<0.001). tGSH and TAS levels were lower in the PP group than in the TPP and C groups (P<0.001), and the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were higher. Furthermore, liver tissue damage, including hemorrhage, hydropic degeneration, and necrosis was observed in the PP group. Administration of taxifolin before pazopanib significantly improved degenerative changes. Our study demonstrated that the administration of taxifolin is significantly effective in preventing pazopanib-induced hepatotoxicity in rats.     

The influence of environmental instability on the reproductive strategy of the critically endangered Acıgöl killifish (Aphanius transgrediens)

To investigate the variability in the reproductive tactics of Acıgöl killifish (Aphanius transgrediens), one of the most seriously threatened freshwater teleost species, this study compared its reproductive ecology in two contrasting habitats that differ substantially in terms of stability of environmental parameters, particularly salinity regime (stable vs. unstable). Fish were sampled monthly from October 2013 to September 2014 with the aim of testing whether the reproductive life-history response of fish to stable and unstable conditions differed. The reproductive effort (gonad weight) of both sexes did not differ significantly between the two habitats, but females in the unstable habitat had significantly lower fecundity and larger eggs. The relationship between fecundity and fish size was stronger in the stable habitat, whereas the relationship was quite variable and uncertain in the unstable habitat. Fish born in the unstable habitat reached their first maturity at a smaller size than those in the stable habitat. The gonado-somatic index and the duration of hydrated eggs showed that reproduction continued from February to May in both habitats; nonetheless, a second spawning event occurred during July and August in the unstable habitat, which included the reproductive contributions of YOY individuals and older generations. This study’s results suggest that A. transgrediens employs varying reproductive strategies against environmental instability in its restricted unique range. This may have further implications for the ways in which habitat-specific conservation methods are used.     

Properties of monocytes generated from haematopoietic CD34+ stem cells from bone marrow of colon cancer patients

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34⁺ stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34⁺ haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14⁺ monocytes were found. They consisted of two subpopulations: CD14⁺⁺CD16⁺ and CD14⁺CD16^?, at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14⁺ monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3⁺ T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu_369–377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8⁺ T lymphocytes (CTL). The CD14⁺⁺CD16⁺ subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34⁺ stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.     

Provider Decisions to Treat Respiratory Illnesses with Antibiotics: Insights from a Randomized Controlled Trial

RationaleLower respiratory tract illness (LRTI) frequently causes adult hospitalization and antibiotic overuse. Procalcitonin (PCT) treatment algorithms have been used successfully in Europe to safely reduce antibiotic use for LRTI but have not been adopted in the United States. We recently performed a feasibility study for a randomized clinical trial (RCT) of PCT and viral testing to guide therapy for non-pneumonic LRTI.ObjectiveThe primary objective of the current study was to understand factors influencing PCT algorithm adherence during the RCT and evaluate factors influencing provider antibiotic prescribing practices for LRTI.ResultsDiagnosis of pneumonia on admission was the only variable significantly associated with non-adherence [7% (adherence) vs. 26% (nonadherence), p = 0.01]. Surveys confirmed possible infiltrate on chest radiograph as important for provider decisions, as were severity of illness, positive sputum culture, abnormal CBC and fever. However, age, patient expectations and medical-legal concerns were also at least somewhat important to prescribing practices. Physician agreement with the importance of viral and PCT testing increased from 42% to 64% (p = 0.007) and 49% to 74% (p = 0.001), respectively, after the study.ConclusionsOptimal algorithm adherence will be important for definitive PCT intervention trials in the US to determine if PCT guided algorithms result in better outcomes than reliance on traditional clinical variables. Factors influencing treatment decisions such as patient age, presence of fever, patient expectations and medical legal concerns may be amenable to education to improve PCT algorithm compliance for LRTI.     

Characterization of the exoskeleton of the Antarctic king crab Paralomis birsteini

Ocean acidification is projected to inhibit the biogenic production of calcium carbonate skeletons in marine organisms. Antarctic waters represent a natural environment in which to examine the long‐term effects of carbonate undersaturation on calcification in marine predators. King crabs (Decapoda: Anomura: Lithodidae), which currently inhabit the undersaturated environment of the continental slope off Antarctica, are potential invasives on the Antarctic shelf as oceanic temperatures rise. Here, we describe the chemical, physical, and mechanical properties of the exoskeleton of the deep‐water Antarctic lithodid Paralomis birsteini and compare our measurements with two decapod species from shallow water at lower latitudes, Callinectes sapidus (Brachyura: Portunidae) and Cancer borealis (Brachyura: Cancridae). In Paralomis birsteini, crabs deposit proportionally more calcium carbonate in their predatory chelae than their protective carapaces, compared with the other two crab species. When exoskeleton thickness and microhardness were compared between the chelae and carapace, the magnitude of the difference between these body regions was significantly greater in P. birsteini than in the other species tested. Hence, there appeared to be a greater disparity in P. birsteini in overall investment in calcium carbonate structures among regions of the exoskeleton. The imperatives of prey consumption and predator avoidance may be influencing the deposition of calcium to different parts of the exoskeleton in lithodids living in an environment undersaturated with respect to calcium carbonate.     

Modern synthetic efforts toward biologically active terpenes

Terpenes represent one of the largest and most diverse classes of secondary metabolites, with over 55,000 members isolated to date. The terpene cyclase enzymes used in nature convert simple, linear hydrocarbon phosphates into an exotic array of chiral, carbocyclic skeletons. Further oxidation and rearrangement results in an almost endless number of conceivable structures. The enormous structural diversity presented by this class of natural products ensures a broad range of biological properties-ranging from anti-cancer and anti-malarial activities to tumor promotion and ion-channel binding. The marked structural differences of terpenes also largely thwart the development of any truly general strategies for their synthetic construction. This review focuses on synthetic strategies directed toward some of the most complex, biologically relevant terpenes prepared by total synthesis within the past decade. Of crucial importance are both the obstacles that modern synthetic chemists must confront when trying to construct such natural products and the key chemical transformations and strategies that have been developed to meet these challenges.