In vitro catabolic effect of protoporphyrin IX in human osteoblast-like cells: possible role of the 18 kDa mitochondrial translocator protein

In several pathological conditions, when conversion of Protoporphyrin (PP)IX into heme is impaired, a toxic accumulation of PPIX might occur. PPIX has been found to have affinity to the mitochondrial Translocator Protein 18 kDa. Since it is known that TSPO is abundant in human osteoblast cells, thus we assumed that PPIX can affect cellular functions via interactions with TSPO in these cells. Therefore we aimed to study the metabolic responses of human osteoblast to a high (10^?5M) concentration of PPIX in vitro. We found that in primary culture of human osteoblast-like cells cell numbers decreased following exposure to PPIX(10^?5M). Cellular [¹⁸F]-FDG incorporation, mitochondrial mass, ATP content were suppressed, and ΔΨm collapsed. Lactate dehydrogenase activity was enhanced in culture media, indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation was not affected. Protein expression of TSPO, VDAC 1, and hexokinase 2 decreased, although the synthesis of mRNA for hexokinase 2 increased. Thus, PPIX(10^?5M) has a cytotoxic effect on human osteoblast-like cell in vitro. Since these cells remain viable following exposure to another TSPO ligand, PK 11195 (10^?5M), as observed previously by us, the mode of action of PPIX on osteoblast-like cells is not identical to that of PK 11195. Accordingly pathological accumulation of PPIX may cause necrosis of osteoblasts leading to bone mass loss. We show that this phenomenon is unrelated to iron overload.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

A novel melatonin agonist Neu-P11 facilitates memory performance and improves cognitive impairment in a rat model of Alzheimer' disease

Previous studies have shown that melatonin is implicated in modulating learning and memory processing. Melatonin also exerts neuroprotective activities against Aβ-induced injury in vitro and in vivo. Neu-P11 (piromelatine, N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-pyran-2-carboxamide) is a novel melatonin (MT1/MT2) receptor agonist and a serotonin 5-HT1A/1D receptor agonist recently developed for the treatment of insomnia. In the present study we firstly investigated whether Neu-P11 and melatonin enhance memory performance in the novel object recognition (NOR) task in rats, and then assessed whether Neu-P11 and melatonin improve neuronal and cognitive impairment in a rat model of Alzheimer' disease (AD) induced by intrahippocampal Aβ_(1–42) injection. The results showed that a single morning or afternoon administration of Neu-P11 enhanced object recognition memory measured at 4 or 24 h after training. Melatonin was effective in the memory facilitating effects only when administered in the afternoon. Further results showed that intrahippocampal Aβ_(1–42) injection resulted in hippocampal cellular loss, as well as decreased learning ability and memory in the Y maze and NOR tasks in rats. Neu-P11 but not melatonin attenuated cellular loss and cognitive impairment in the rat AD model. The current data suggest that Neu-P11 may serve as a novel agent for the treatment of AD.     

DNA Methylation, Behavior and Early Life Adversity

The impact of early physical and social environments on life-long phenotypes is well known. Moreover, we have documented evidence for gene–environment interactions where identical gene variants are associated with different phenotypes that are dependent on early life adversity. What are the mechanisms that embed these early life experiences in the genome? DNA methylation is an enzymatically-catalyzed modification of DNA that serves as a mechanism by which similar sequences acquire cell type identity during cellular differentiation and embryogenesis in the same individual. The hypothesis that will be discussed here proposes that the same mechanism confers environmental-exposure specific identity upon DNA providing a mechanism for embedding environmental experiences in the genome, thus affecting long-term phenotypes. Particularly important is the environment early in life including both the prenatal and postnatal social environments.     

Oxygen Mask Related Nasal Integument and Osteocartilagenous Disorders in F-16 Fighter Pilots

Background

A preliminary survey showed half of the participating Royal Netherlands Air Force (RNLAF) F-16 fighter pilots to have nasal integument and osteocartilagenous disorders related to wearing in-flight oxygen masks.

Aim

To make an inventory of these disorders and possible associated factors.

Methods

All RNLAF F-16 pilots were requested to fill out a semi-structured questionnaire for a cross-sectional survey. Additionally, one squadron in The Netherlands and pilots in operational theater were asked to participate in a prospective study that required filling out a pain score after each flight. Pilot- and flight-related variables on all participants were collected from the RNLAF database. A linear mixed model was built to identify associated factors with the post-flight pain score.

Results

The response rate to the survey was 83%. Ninety of the 108 participants (88%, 6 missing) reported tenderness, irritation, pain, erythema, skin lesions, callous skin, or swelling of nasal bridge integument or architecture. Seventy-two participants (71%, 6 missing) reported their symptoms to be troublesome after a mean of 6±3 out of 10 flights (0;10, 54 missing). Sixty-six pilots participated in scoring post-flight pain. Pain scores were significantly higher if a participant had ≥3 nasal disorders, after longer than average flights, after flying abroad, and after flying with night vision goggles (respectively +2.7 points, p = 0.003; +0.2 points, p = 0.027; +1.8 points, p = 0.001; +1.2 points p = 0.005). Longer than average NVG flights and more than average NVG hours per annum decreased painscores (respectively −0.8 points, p = 0.017; −0.04 points, p = 0.005).

Conclusions

The majority of the RNLAF F-16 fighter pilot community has nasal disorders in the contact area of the oxygen mask, including pain. Six pilot- or flight-related characteristics influence the experienced level of pain.     

A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis.     

Finding a common motif of RNA sequences using genetic programming: the GeRNAMo system

We focus on finding a consensus motif of a set of homologous or functionally related RNA molecules. Recent approaches to this problem have been limited to simple motifs, require sequence alignment, and make prior assumptions concerning the data set. We use genetic programming to predict RNA consensus motifs based solely on the data set. Our system -- dubbed GeRNAMo (Genetic programming of RNA Motifs) -- predicts the most common motifs without sequence alignment and is capable of dealing with any motif size. Our program only requires the maximum number of stems in the motif, and if prior knowledge is available the user can specify other attributes of the motif (e.g., the range of the motif's minimum and maximum sizes), thereby increasing both sensitivity and speed. We describe several experiments using either ferritin iron response element (IRE); signal recognition particle (SRP); or microRNA sequences, showing that the most common motif is found repeatedly, and that our system offers substantial advantages over previous methods.