Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.     

Rufloxacin induced photosensitization in bio-models of increasing complexity.

Rufloxacin belongs to the class of fluoroquinolones that act mainly as specific inhibitors of bacterial Topoisomerase II. These drugs are widely known to be involved in various diseases ranging from cutaneous reactions to aging. The type II photosensitizing activity of Rufloxacin has been already demonstrated on calf thymus DNA and free nucleosides. The aim of this study is to examine in control untreated and UVA irradiated human fibroblasts the modifications on DNA status induced by Rufloxacin added in the culture medium. This allows to investigate the photosensitizing activity of Rufloxacin in a more complex cell model. Fibroblasts, either in the presence or in the absence of Rufloxacin, were exposed to UVA irradiation for different times. An experimental protocol was followed in order to evaluate the amount of single-strand breaks (SSB) and double-strand breaks (DSB) DNA fragmentation by comet assay, and plasmid photocleavage. The presence of oxidized bases was also evaluated using the 8-OH-dGuo test. The comet assay test was also employed to assess cellular repair capacity. The intracellular drug concentration was verified by HPLC-MS. The results confirming the role of Rufloxacin as photosensitizer were: (i) a time-dependent increase in DNA fragmentation when fibroblasts were irradiated in the presence of Rufloxacin; (ii) the efficiency of the cellular repair machinery to be exhaustive after 2 h (whereas no correlation between irradiation time and DNA damage repair was observed with a higher level of DNA fragmentation after shorter irradiation times); (iii) the increased number of cells exhibiting high DNA fragmentation, seen as comets with long tails, was not accompanied by a similar large extent of oxidised DNA base formation, as measured by 8-OH-dGuo analysis; (iv) the double helix SSB, formed in plasmid photosensitization, agreed with the comet assay results, pointing out a good correlation among the cell system and the simpler models used.     

Absolute configuration of the isomer of fluorocitrate that inhibits aconitase

The absolute configuration of the isomer of fluorocitrate that is a strong inhibitor and activator of aconitase has been determined to be 2R, 3R, as shown in the Fischer and perspective diagrams below.

Fischer diagram Perspective diagramThis is the isomer described by Dummel and Kun (1969, J. Biol. Chem.244, 2966) and defined as (?)-erythro-fluorocitrate by them. We report here structural studies of the mirror image (enantiomer) of this isomer. Crystals of the complex of the diethyl ester of (+)-erythro-fluorocitrate with (?)-methylbenzylamine were studied by X-ray diffraction techniques. Since the absolute configuration of (?)-methylbenzylamine has already been determined experimentally, the absolute configuration of the (+)-erythro isomer of fluorocitrate is thereby established. This isomer is shown to be noninhibitory with the enzyme aconitase, while its racemate is a powerful inhibitor. Thus it is proved that the absolute configuration of the isomer of fluorocitrate that is formed from fluoroacetyl-CoA by the enzyme citrate synthase, and that inhibits aconitase, is the 2R, 3R, isomer.     

Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples

The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples. The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection. The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods. The MicroFoss system detected Listeria spp. in 12 samples, which were not detected by culture, and the culture method detected Listeria spp. in 15 samples, which were not detected by the MicroFoss method. This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods. The specificity value determined for the MicroFoss system was 92.7%. The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features. Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L. monocytogenes being the most predominant isolate detected. The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples. The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.     

Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca²⁺-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca²⁺ perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca²⁺. These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.     

Aminosuberoyl hydroxamic acids (ASHAs): a potent new class of HDAC inhibitors

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are currently in advanced clinical trials for the treatment of cancer. Vorinostat (Zolinza, SAHA) is a hydroxamic acid approved for the treatment of patients with cutaneous T-cell lymphoma who have progressive, persistent or recurrent disease on or following two systemic therapies. As part of an on-going effort to better understand the nature of the HDAC enzyme/inhibitor interaction and design highly effective HDAC inhibitors, we herein report the design, synthesis and HDAC inhibitory activity of a vorinostat-derived series of substrate-based HDAC inhibitors.     

Mesoglycan induces keratinocyte activation by triggering syndecan-4 pathway and the formation of the annexin A1/S100A11 complex

Wound healing is a dynamic process comprising multiple events, such as inflammation, re-epithelialization, and tissue remodeling. Re-epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re-epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non-healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re-epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan-4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro-healing drug for skin wound care.     

A computational index derived from whole-genome copy number analysis is a novel tool for prognosis in early stage lung squamous cell carcinoma

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.     

Does medio-lateral motion occur in the normal knee? An in-vitro study in passive motion

Medio-lateral translation during knee flexion continues to raise controversy. Small population sizes, small joint flexion ranges, less-reliable measurement techniques and disparate experimental conditions led to inconsistent reports in the past. To study this subject with more accurate and reliable measurements, we carried out femur and tibia tracking in 22 intact cadaver knees during passive joint motion using a state-of-the-art surgical navigation system. Trackers with active light-emitting diodes were fixed onto the femur and tibia, and an instrumented pointer was used to digitize a number of anatomical landmarks. International recommendations were adopted for anatomical-based reference frame definitions and joint kinematic analysis. For the first time, knee joint translations were reported in both the femoral and tibial reference frames, and over a flexion/extension arc as large as 140°. During flexion, in the femoral reference frame, the center of the tibial plateau moved 4.8 ± 2.8mm medially when averaged over the specimens. In the tibial frame, the knee center moved 13.3 ± 5.7 mm laterally. The relative femoral-to-tibial medio-lateral translation was, on average over the specimens, nearly 20% of the width of the tibial plateau, and can be as large as 35%. Medio-lateral translation occurs in the natural normal knee joint.