Airborne urban particles (Milan winter-PM2.5) cause mitotic arrest and cell death: Effects on DNA, mitochondria, AhR binding and spindle organization

Airborne particulate matter (PM) is considered to be an important contributor to lung diseases. In the present study we report that Milan winter-PM2.5 inhibited proliferation in human bronchial epithelial cells (BEAS-2B) by inducing mitotic arrest. The cell cycle arrest was followed by an increase in mitotic-apoptotic cells, mitotic slippage and finally an increase in "classical" apoptotic cells. Exposure to winter-PM10 induced only a slight effect which may be due to the presence of PM2.5 in this fraction while pure combustion particles failed to disturb mitosis. Fewer cells expressing the mitosis marker phospho-histone H3 compared to cells with condensed chromosomes, suggest that PM2.5 induced premature mitosis. PM2.5 was internalized into the cells and often localized in laminar organelles, although particles without apparent plasma membrane covering were also seen. In PM-containing cells mitochondria and lysosomes were often damaged, and in mitotic cells fragmented chromosomes often appeared. PM2.5 induced DNA strands breaks and triggered a DNA-damage response characterized by increased phosphorylation of ATM, Chk2 and H2AX; as well as induced a marked increase in expression of the aryl hydrocarbon receptor (AhR)-regulated genes, CYP1A1, CYP1B1 and AhRR. Furthermore, some disturbance of the organization of microtubules was indicated. It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.     

A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB

The multicopy sRNA LhrC of the intracellular pathogen Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.     

Risk of renal stone events in primary hyperparathyroidism before and after parathyroid surgery: controlled retrospective follow up study

AimTo study the risk of renal stone episodes and risk factors for renal stones in primary hyperparathyroidism before and after surgery.DesignRegister based, controlled retrospective follow up study.SettingTertiary hospitals in Denmark.Participants674 consecutive patients with surgically verified primary hyperparathyroidism. Each patient was compared with three age- and sex-matched controls randomly drawn from the background population. Hospital admissions for renal stone disease were compared between patients and controls. Risk factors for renal stones among patients were assessed.ResultsRelative risk of a stone episode was 40 (95% confidence interval 31 to 53) before surgery and 16 (12 to 23) after surgery. Risk was increased 10 years before surgery, and became normal more than 10 years after surgery. Stone-free survival 20 years after surgery was 90.4% in patients and 98.7% in controls (risk difference 8.3%, 4.8% to 11.7%). Patients with preoperative stones had 27 times the risk of postoperative stone incidents than controls. Before surgery, males had more stone episodes than females and younger patients had more stone episodes than older patients. Neither parathyroid pathology, weight of removed tissue, plasma calcium levels, nor skeletal pathology (fractures) influenced the risk of renal stones. After surgery, younger age, preoperative stones and ureteral strictures were significant risk factors for stones.ConclusionsThe risk of renal stones is increased in primary hyperparathyroidism and decreases after surgery. The risk profile is normal 10 years after surgery. Preoperative stone events increase the risk of postoperative stones. Stone formers and non-stone formers had the same risk of skeletal complications.

What is already known on this topic

Patients with primary hyperparathyroidism have an increased risk of renal stone eventsThe extent to which parathyroid surgery reduces the risk of further stones is unclear

What this study adds

The risk of a new stone event was 8.3% higher in patients than in controls after surgeryIn patients with stone disease before operation the risk rate for a postoperative stone event was 27times that in controlsThe risk of a renal stone event was higher than the risk among controls until more than 10 years after surgery     

Rational combinatorial chemistry-based selection,synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.     

ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.     

Outside St. Jørgen: leprosy in the medieval Danish city of Odense

Leprosy was a common and dreaded disease in the Danish Middle Ages (AD 1050-1536). Starting in the second half of the 13th century, leprosaria were established in many Danish towns and cities. In the city of Odense (on the island of Funen, Denmark), the cemetery of the leprosarium was totally excavated, and four nonleprosarium medieval and early modern cemeteries have been partly excavated. This paper explores the frequency of leprosy in the nonleprosarium cemeteries in Odense, and looks for evidence of selective exclusion from the ordinary population. The analyses are based on 733 skeletons from four cemeteries in Odense: the Gray Friars monastery, St. Albani parish church, St. Knuds cathedral, and Black Friars monastery. Seven lesions are scored and, based on known epidemiological properties (i.e., specificity and sensitivity) of these lesions, scores were transformed to statistics characterizing an individual's risk of having suffered from leprosy. This statistical approach remains of primary theoretical value, pending confirmation by independent research groups at other sites. Prevalence of the skeletal manifestation of leprosy at death varied between 0-17% among the different cemeteries in Odense. The highest prevalence was seen in cemeteries with many burials before AD 1400. It is estimated that before AD 1400, between 14-17% of those buried in the nonleprosarium cemeteries suffered from leprosy. In all nonleprosarium cemeteries, there was evidence for selective exclusion of people with facial leprosy lesions. For a short period just up to AD 1300, the cemetery of the Odense leprosarium had, on average, more than 20 yearly burials. The establishment of the leprosarium was followed within a relatively short period by a dramatic decline in the number of sufferers of leprosy in the nonleprosarium cemeteries. The number of yearly burials in the leprosarium cemetery also declined rapidly during the 14th century. The present analyses do not permit conclusions about the reasons for this decline in leprosy prevalence.     

Structural divergence between the two subgroups of P5 ATPases

Evolution of P5 type ATPases marks the origin of eukaryotes but still they remain the least characterized pumps in the superfamily of P-type ATPases. Phylogenetic analysis of available sequences suggests that P5 ATPases should be divided into at least two subgroups, P5A and P5B. P5A ATPases have been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify differences in the primary sequences between the two subgroups. P5A and P5B ATPases appear have a very different membrane topology from other P-type ATPases with two and one, respectively, additional transmembrane segments inserted in the N-terminal end. Based on conservation of residues in the transmembrane region, the two P5 subgroups most likely have different substrate specificities although these cannot be predicted from their sequences. Furthermore, sequence differences between P5A and P5B ATPases are identified in the catalytic domains that could influence key kinetic properties differentially. Together these findings indicate that P5A and P5B ATPases are structurally and functionally different.     

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity.