Angiogenesis and angioregression gene expression analyses in swine corpus luteum

The corpus luteum (CL) lifespan is characterized by a rapid growth, differentiation and controlled regression of the luteal tissue, accompanied by an intense angiogenesis and angioregression. Indeed, the CL is one of the most highly vascularised tissue in the body with a proliferation rate of the endothelial cells 4- to 20-fold more intense than in some of the most malignant human tumours. This angiogenic process should be rigorously controlled to allow the repeated opportunities of fertilization. After a first period of rapid growth, the tissue becomes stably organized and prepares itself to switch to the phenotype required for its next apoptotic regression. In pregnant swine, the lifespan of the CLs must be extended to support embryonic and foetal development and vascularisation is necessary for the maintenance of luteal function. Among the molecules involved in the angiogenesis, Vascular Endothelial Growth Factor (VEGF) is the main regulator, promoting endothelial cells proliferation, differentiation and survival as well as vascular permeability and vessel lumen formation. During vascular invasion and apoptosis process, the remodelling of the extracellular matrix is essential for the correct evolution of the CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). Another important factor that plays a role in the processes of angiogenesis and angioregression during the CL formation and luteolysis is the isopeptide Endothelin-1 (ET-1), which is well-known to be a potent vasoconstrictor and mitogen for endothelial cells. The goal of the present thesis was to study the role and regulation of vascularisation in an adult vascular bed. For this purpose, using a precisely controlled in vivo model of swine CL development and regression, we determined the levels of expression of the members of VEGF system (VEGF total and specific isoforms; VEGF receptor-1, VEGFR-1; VEGF receptor-2, VEGFR-2) and ET- 1 system (ET-1; endothelin converting enzyme-1, ECE-1; endothelin receptor type A, ET-A) as well as the activity of the Ca++/Mg++-dependent endonucleases and gelatinases (MMP-2 and MMP-9). Three experiments were conducted to reach such objectives in CLs isolated from ovaries of cyclic, pregnant or fasted gilts. In the Experiment I, we evaluated the influence of acute fasting on VEGF production and VEGF, VEGFR-2, ET-1, ECE-1 and ET-A mRNA expressions in CLs collected on day 6 after ovulation (midluteal phase). The results indicated a down-regulation of VEGF, VEGFR-2, ET-1 and ECE-1 mRNA expression, although no change was observed for VEGF protein. Furthermore, we observed that fasting stimulated steroidogenesis by luteal cells. On the basis of the main effects of VEGF (stimulation of vessel growth and endothelial permeability) and ET-1 (stimulation of endothelial cell proliferation and vasoconstriction, as well as VEGF stimulation), we concluded that feed restriction possibly inhibited luteal vessel development. This could be, at least in part, compensated by a decrease of vasal tone due to a diminution of ET-1, thus ensuring an adequate blood flow and the production of steroids by the luteal cells. In the Experiment II, we investigated the relationship between VEGF, gelatinases and Ca++/Mg++-dependent endonucleases activities with the functional CL stage throughout the oestrous cycle and at pregnancy. The results demonstrated differential patterns of expression of those molecules in correspondence to the different phases of the oestrous cycle. Immediately after ovulation, VEGF mRNA/protein levels and MMP-9 activity are maximal. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed. These results suggested that during the very early luteal phase, high MMPs activities coupled with high VEGF levels drive the tissue to an angiogenic phenotype, allowing CL growth under LH (Luteinising Hormone) stimulus, while during the late luteal phase, low VEGF and elevate MMPs levels may play a role in the apoptotic tissue and extracellular matrix remodelling during structural luteolysis. In the Experiment III, we described the expression patterns of all distinct VEGF isoforms throughout the oestrous cycle. Furthermore, the mRNA expression and protein levels of both VEGF receptors were also evaluated. Four novel VEGF isoforms (VEGF144, VEGF147, VEGF182, and VEGF164b) were found for the first time in swine and the seven identified isoforms presented four different patterns of expression. All isoforms showed their highest mRNA levels in newly formed CLs (day 1), followed by a decrease during mid-late luteal phase (days 10–17), except for VEGF182, VEGF188 and VEGF144 that showed a differential regulation during late luteal phase (day 14) or at luteolysis (day 17). VEGF protein levels paralleled the most expressed and secreted VEGF120 and VEGF164 isoforms. The VEGF receptors mRNAs showed a different pattern of expression in relation to their ligands, increasing between day 1 and 3 and gradually decreasing during the mid-late luteal phase. The differential regulation of some VEGF isoforms principally during the late luteal phase and luteolysis suggested a specific role of VEGF during tissue remodelling process that occurs either for CL maintenance in case of pregnancy or for noncapillary vessel development essential for tissue removal during structural luteolysis. In summary, our findings allow us to determine relationships among factors involved in the angiogenesis and angioregression mechanisms that take place during the formation and regression of the CL. Thus, CL provides a very interesting model for studying such factors in different fields of the basic research.

Dati geochimici ed ecologici di foraminiferi planctonici. Implicazioni paleoceanografiche in due aree chiave: Oceano Meridionale e Mare Mediterraneo.

La studio della distribuzione spaziale e temporale della associazioni a foraminiferi planctonici, campionati in zone con differente regime idrografico, ha permesso di comprendere che molte specie possono essere diagnostiche della presenza di diverse masse d’acqua superficiali e sottosuperficiali e di diversi regimi di nutrienti nelle acque oceaniche. Parte di questo lavoro di tesi si basa sullo studio delle associazioni a foraminiferi planctonici attualmente viventi nel Settore Pacifico dell’Oceano Meridionale (Mare di Ross e Zona del Fronte Polare) e nel Mare Mediterraneo (Mar Tirreno Meridionale). L’obiettivo di questo studio è quello di comprendere i fattori (temperatura, salinità, nutrienti etc.) che determinano la distribuzione attuale delle diverse specie al fine di valutarne il valore di “indicatori” (proxies) utili alla ricostruzione degli scenari paleoclimatici e paleoceanografici succedutisi in queste aree. I risultati documentano che la distribuzione delle diverse specie, il numero di individui e le variazioni nella morfologia di alcuni taxa sono correlate alle caratteristiche chimico-fisiche della colonna e alla disponibilità di nutrienti e di clorofilla. La seconda parte del lavoro di tesi ha previsto l’analisi degli isotopi stabili dell’ossigeno e del rapporto Mg/Ca in gusci di N. pachyderma (sin) prelevati da pescate di micro zooplancton (per tarare l’equazione di paleo temperatura) da un box core e da una carota provenienti dalla zona del Fronte Polare (Oceano Pacifico meridionale), al fine di ricostruire le variazioni di temperatura negli ultimi 13 ka e durante la Mid-Pleistocene Revolution. Le temperature, dedotte tramite i valori degli isotopi stabili dell’ossigeno, sono coerenti con le temperature attuali documentate in questa zona e il trend di temperatura è paragonabile a quelli riportati in letteratura anche per eventi climatici come lo Younger Dryas e il mid-Holocene Optimum. I valori del rapporto Mg/Ca misurato tramite due diverse tecniche di analisi (laser ablation e analisi in soluzione) sono risultati sempre molto più alti dei valori riportati in letteratura per la stessa specie. La laser ablation sembra carente dal punto di vista del cleaning del campione e da questo studio emerge che le due tecniche non sono comparabili e che non possono essere usate indifferentemente sullo stesso campione. Per quanto riguarda l’analisi dei campioni in soluzione è stato migliorato il protocollo di cleaning per il trattamento di campioni antartici, che ha permesso di ottenere valori veritieri e utili ai fini delle ricostruzioni di paleotemperatura. Tuttavia, rimane verosimile l’ipotesi che in ambienti particolari come questo, con salinità e temperature molto basse, l’incorporazione del Mg all’interno del guscio risenta delle condizioni particolari e che non segua quindi la relazione esponenziale con la temperatura ampiamente dimostrata ad altre latitudini.

Evidenze micropaleontologiche e sedimentologiche di cicli deposizionali e climatici circa millenari nei Depositi Tardoquaternari della Piana dell'Arno e del Delta del Po

A multidisciplinary study was carried out on the Late Quaternary-Holocene subsurface deposits of two Mediterranean coastal areas: Arno coastal plain (Northern Tyrrhenian Sea) and Modern Po Delta (Northern Adriatic Sea). Detailed facies analyses, including sedimentological and micropalaeontological (benthic foraminifers and ostracods) investigations, were performed on nine continuously-cored boreholes of variable depth (ca. from 30 meters to100 meters). Six cores were located in the Arno coastal plain and three cores in the Modern Po Delta. To provide an accurate chronological framework, twenty-four organic-rich samples were collected along the fossil successions for radiocarbon dating (AMS 14C). In order to reconstruct the depositional and palaeoenvironmental evolution of the study areas, core data were combined with selected well logs, provided by local companies, along several stratigraphic sections. These sections revealed the presence of a transgressive-regressive (T-R) sequence, composing of continental, coastal and shallow-marine deposits dated to the Late Pleistocene-Holocene period, beneath the Arno coastal plain and the Modern Po Delta. Above the alluvial deposits attributed to the last glacial period, the post-glacial transgressive succession (TST) consists of back-barrier, transgressive barrier and inner shelf deposits. Peak of transgression (MFS) took place around the Late-Middle Holocene transition and was identified by subtle micropalaeontological indicators within undifferentiated fine-grained deposits. Upward a thick prograding succession (HST) records the turnaround to regressive conditions that led to a rapid delta progradation in both study areas. Particularly, the outbuilding of modern-age Po Delta coincides with mud-belt formation during the late HST (ca. 600 cal yr BP), as evidenced by a fossil microfauna similar to the foraminiferal assemblage observed in the present Northern Adriatic mud-belt. A complex interaction between allocyclic and autocyclic factors controlled facies evolution during the highstand period. The presence of local parameters and the absence of a predominant factor prevent from discerning or quantifying consequences of the complex relationships between climate and deltaic evolution. On the contrary transgressive sedimentation seems to be mainly controlled by two allocyclic key factors, sea-level rise and climate variability, that minimized the effects of local parameters on coastal palaeoenvironments. TST depositional architecture recorded in both study areas reflects a well-known millennial-scale variability of sea-level rising trend and climate during the Late glacial-Holocene period. Repeated phases of backswamp development and infilling by crevasse processes (parasequences) were recorded in the subsurface of Modern Po Delta during the early stages of transgression (ca. 11,000-9,500 cal yr BP). In the Arno coastal plain the presence of a deep-incised valley system, probably formed at OSI 3/2 transition, led to the development of a thick (ca. 35-40 m) transgressive succession composed of coastal plain, bay-head delta and estuarine deposits dated to the Last glacial-Early Holocene period. Within the transgressive valley fill sequence, high-resolution facies analyses allowed the identification and lateral tracing of three parasequences of millennial duration. The parasequences, ca. 8-12 meters thick, are bounded by flooding surfaces and show a typical internal shallowing-upward trend evidenced by subtle micropalaeontological investigations. The vertical stacking pattern of parasequences shows a close affinity with the step-like sea-level rising trend occurred between 14,000-8,000 cal years BP. Episodes of rapid sea-level rise and subsequent stillstand phases were paralleled by changes in climatic conditions, as suggested by pollen analyses performed on a core drilled in the proximal section of the Arno palaeovalley (pollen analyses performed by Dr. Marianna Ricci Lucchi). Rapid shifts to warmer climate conditions accompanied episodes of rapid sea-level rise, in contrast stillstand phases occurred during temporary colder climate conditions. For the first time the palaeoclimatic signature of high frequency depositional cycles is clearly documented. Moreover, two of the three "regressive" pulsations, recorded at the top of parasequences by episodes of partial estuary infilling in the proximal and central portions of Arno palaeovalley, may be correlated with the most important cold events of the post-glacial period: Younger Dryas and 8,200 cal yr BP event. The stratigraphic and palaeoclimatic data of Arno coastal plain and Po Delta were compared with those reported for the most important deltaic and coastal systems in the worldwide literature. The depositional architecture of transgressive successions reflects the strong influence of millennial-scale eustatic and climatic variability on worldwide coastal sedimentation during the Late glacial-Holocene period (ca. 14,000-7,000 cal yr BP). The most complete and accurate record of high-frequency eustatic and climatic events are usually found within the transgressive succession of very high accommodation settings, such as incised-valley systems where exceptionally thick packages of Late glacial-Early Holocene deposits are preserved.

Pyrochlore der Pegmatit-Provinz Nazareno, Brasilien

Zusammenfassung:Mit Hilfe einer neuen Formel für die Minerale der Pyrochlor-Gruppe werden sämtliche Endglieder der Na-Ca-Mikrolithe und der Ba-haltigen Mikrolithe aus der Pegmatit-Provinz Nazareno beschrieben: Die Na-reichsten Proben haben nahezu die Idealzusammensetzung eines idealen Pyrochlors, d.h. . Die Ca-reichsten Varietäten weisen maximal auf, wobei der Besetzungsanteil des Ca am A2+ ca. 93% beträgt. Die Ba-haltigen Mikrolithe sind durch eine Defektstruktur gekennzeichnet, wobei für das mögliche Endglied kein Beispiel in den Daten vorliegt. Das Endglied mit dem geringsten Defektcharakter hat folgende Stöchiometrie:

Einfluss moderater intraischämischer Hypothermie auf die Proliferation und Differenzierung neuronaler Stammzellen 28 Tage nach inkompletter Vorderhirnischämie und Reperfusion der adulten Ratte

Hypothermie schützt Neurone vor hypoxischen, ischämischen und traumatischen Schädigungen. Bisher ist jedoch unklar, ob Hypothermie auch endogene Reparaturmechanismen beeinflusst. Die vorliegende Arbeit untersucht daher den Einfluss intraischämischer Hypothermie auf das neuroregenerative Potential des Gehirns nach zerebraler Ischämie.rn50 männliche Sprague-Dawley Ratten wurden hierzu anästhesiert, intubiert und in folgende Versuchsgruppen randomisiert: Normotherme Ischämie (Normo/BACO), intraischämische Hypothermie (Hypo/BACO) sowie korrespondierende scheinoperierte Kontrollgruppen (Normo/Sham und Hypo/Sham). In den Gruppen Normo/Sham und Normo/BACO wurde die perikranielle Temperatur konstant bei 37 °C gehalten während sie in den Gruppen Hypo/Sham und Hypo/BACO für 85 min auf 33 °C gesenkt wurde. Durch bilaterale Okklusion der Aa. carotides communes in Kombination mit hämorrhagischer Hypotension wurde in BACO-Tieren eine 14-minütige inkomplette globale zerebrale Ischämie induziert. Tiere der Kontroll-Gruppen (Sham) blieben ohne Induktion einer Ischämie in Narkose. 15 weitere Tiere durchliefen nicht den operativen Versuchsteil und bildeten die Nativ-Gruppe, die als Referenz für die natürliche Neurogenese diente. Zur in-vivo-Markierung der Stammzellen wurde vom ersten bis siebten postoperativen Tag Bromodeoxyurindine (BrdU) injiziert. Nach 28 Tagen wurden die Gehirne entnommen. Die Analyse des histopathologischen Schadens erfolgte anhand HE-gefärbter Hirnschnitte, die Quantifikation der absoluten Anzahl neu gebildeter Zellen im Gyrus dentatus erfolgte mittels BrdU-Färbung. Anhand einer BrdU/NeuN-Immunfluoreszenz-Doppelfärbung konnte der Anteil neu generierter Neurone bestimmt werden.rnNach zerebraler Ischämie zeigten Tiere mit Normothermie eine Schädigung der CA 1-Region von über 50 % während hypotherme Ischämietiere einen Schaden von weniger als 10 % aufwiesen. Tiere ohne Ischämie (Hypo/Sham, Normo/Sham, Nativ) zeigten keinen histopathologischen Schaden. Die Anzahl neu gebildeter Neurone im Gyrus dentatus lag für normotherme Ischämietiere (Normo/BACO) bei 18819 und für Tiere mit intraischämischer Hypothermie (Hypo/BACO) bei 15175 neuen Neuronen. In den Kontroll-Gruppen wiesen Tiere der Gruppe Normo/Sham 5501, Tiere der Gruppe Hypo/Sham 4600 und Tiere der Nativ-Gruppe 5974 neu generierte Neurone auf.rnDiese Daten bestätigen frühere Studien, die eine Reduktion des neuronalen Schadens durch intraischämische Hypothermie zeigten. Infolge des ischämischen Stimulus kam es im Vergleich zu beiden Kontroll- und der Nativ-Gruppe zu einem signifikanten Anstieg der Anzahl neuer Neurone in beiden Ischämiegruppen unabhängig von der Temperatur. Somit scheint das Ausmaß der histopathologischen Schädigung keinen Einfluss auf die Anzahl neu gebildeter Neurone zu haben. Darüber hinaus beeinflusste die therapeutische Hypothermie auch nicht die natürliche Neurogeneserate. Die erhobenen Daten lassen vermuten, dass Hypothermie keinen Effekt auf die Anzahl und Differenzierung neuronaler Stammzellen aufweist, unabhängig davon, ob eine zerebrale Schädigung vorliegt.

Radiation-induced defects in calcium fluoride and their influence on material properties under 193 nm laser irradiation

Calcium fluoride (CaF2) is one of the key lens materials in deep-ultraviolet microlithography because of its transparency at 193 nm and its nearly perfect optical isotropy. Its physical and chemical properties make it applicable for lens fabrication. The key feature of CaF2 is its extreme laser stability. rnAfter exposing CaF2 to 193 nm laser irradiation at high fluences, a loss in optical performance is observed, which is related to radiation-induced defect structures in the material. The initial rapid damage process is well understood as the formation of radiation-induced point defects, however, after a long irradiation time of up to 2 months, permanent damage of the crystals is observed. Based on experimental results, these permanent radiation-induced defect structures are identified as metallic Ca colloids.rnThe properties of point defects in CaF2 and their stabilization in the crystal bulk are calculated with density functional theory (DFT). Because the stabilization of the point defects and the formation of metallic Ca colloids are diffusion-driven processes, the diffusion coefficients for the vacancy (F center) and the interstitial (H center) in CaF2 are determined with the nudged elastic band method. The optical properties of Ca colloids in CaF2 are obtained from Mie-theory, and their formation energy is determined.rnBased on experimental observations and the theoretical description of radiation-induced point defects and defect structures, a diffusion-based model for laser-induced material damage in CaF2 is proposed, which also includes a mechanism for annealing of laser damage. rn

Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells

Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca(2+) and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.

Plasma membrane repair and cellular damage control: the annexin survival kit

Plasmalemmal injury is a frequent event in the life of a cell. Physical disruption of the plasma membrane is common in cells that operate under conditions of mechanical stress. The permeability barrier can also be breached by chemical means: pathogens gain access to host cells by secreting pore-forming toxins and phospholipases, and the host's own immune system employs pore-forming proteins to eliminate both pathogens and the pathogen-invaded cells. In all cases, the influx of extracellular Ca(2+) is being sensed and interpreted as an "immediate danger" signal. Various Ca(2+)-dependent mechanisms are employed to enable plasma membrane repair. Extensively damaged regions of the plasma membrane can be patched with internal membranes delivered to the cell surface by exocytosis. Nucleated cells are capable of resealing their injured plasmalemma by endocytosis of the permeabilized site. Likewise, the shedding of membrane microparticles is thought to be involved in the physical elimination of pores. Membrane blebbing is a further damage-control mechanism, which is triggered after initial attempts at plasmalemmal resealing have failed. The members of the annexin protein family are ubiquitously expressed and function as intracellular Ca(2+) sensors. Most cells contain multiple annexins, which interact with distinct plasma membrane regions promoting membrane segregation, membrane fusion and--in combination with their individual Ca(2+)-sensitivity--allow spatially confined, graded responses to membrane injury.

Calcium-modulated chloride pathways contribute to chloride flux in murine cystic fibrosis-affected macrophages

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.

Nasolabial symmetry and aesthetics in children with complete unilateral cleft lip and palate

The objective of this study was to evaluate the association between nasolabial symmetry and aesthetics in children with complete unilateral cleft lip and palate (CUCLP). Frontal and basal photographs of 60 consecutively treated children with CUCLP (cleft group: 41 boys and 19 girls, mean (SD) age 11 (2) years) and 44 children without clefts (control group: 16 boys and 28 girls, mean (SD) age 11(2) years), were used for evaluation of nasolabial symmetry and aesthetics. Nasal and labial measurements were made to calculate the coefficient of asymmetry (CA). The 5-grade aesthetic index described by Asher-McDade et al. was used to evaluate nasolabial appearance. Correlation and regression analysis were used to identify an association between aesthetics and CA, sex, and the presence of CUCLP. Ten measurements in the cleft, and 2 in the control, group differed significantly between the cleft and non-cleft (or right and left) sides, respectively. The significantly higher values of 9 of 11 CA in the children with CUCLP indicated that they had more asymmetrical nasolabial areas than children without clefts. However, the regression analyses showed that only a few CA were associated with nasolabial aesthetics. In conclusion, nasolabial aesthetics and nasolabial symmetry seem to be only weakly associated in patients with CUCLP.

Factors Influencing Spatial Variability in Nitrogen Processing in Nitrogen-Saturated Soils

Nitrogen (N) saturation is an environmental concern for forests in the eastern U.S. Although several watersheds of the Fernow Experimental Forest (FEF), West Virginia exhibit symptoms of Nsaturation, many watersheds display a high degree of spatial variability in soil N processing. This study examined the effects of temperature on net N mineralization and nitrification in N-saturatedsoils from FEF, and how these effects varied between high N-processing vs. low N-processingsoils collected from two watersheds, WS3 (fertilized with [NH4]2SO4) and WS4 (untreated control). Samples of forest floor material (O2 horizon) and mineral soil (to a 5-cm depth) were taken from three subplots within each of four plots that represented the extremes of highest and lowest ratesof net N mineralization and nitrification (hereafter, high N and low N, respectively) of untreated WS4 and N-treated WS3: control/low N, control/high N, N-treated/low N, N-treated/high N. Forest floor material was analyzed for carbon (C), lignin,and N. Subsamples of mineral soil were extractedimmediately with 1 N KCl and analyzed for NH4+and NO3– to determine preincubation levels. Extracts were also analyzed for Mg, Ca, Al, and pH. To test the hypothesis that the lack of net nitrification observed in field incubations on the untreated/low N plot was the result of absence ofnitrifier populations, we characterized the bacterial community involved in N cycling by amplification of amoA genes. Remaining soil was incubated for 28 d at three temperatures (10, 20, and30°C), followed by 1 N KCl extraction and analysis for NH4+ and NO3–. Net nitrification was essentially 100% of net N mineralization for all samples combined. Nitrification rates from lab incubation sat all temperatures supported earlier observations based on field incubations. At 30°C, rates from N- t reated/high N were three times those of N-treated/low N. Highest rates were found for untreated/high N (two times greater than those of N-treated/high N), whereas untreated/low N exhibited no net nitrification. However, soils exhibitingno net nitrification tested positive for presence of nitrifying bacteria, causing us to reject our initial hypothesis. We hypothesize that nitrifier populations in such soil are being inhibited by a combination of low Ca:Al ratios in mineral soil and allelopathic interactions with mycorrhizae of ericaceous species in the herbaceous layer.

Calcium waves driven by "sensitization" wave-fronts

OBJECTIVE: Cellular Ca(2+) waves are understood as reaction-diffusion systems sustained by Ca(2+)-induced Ca(2+) release (CICR) from Ca(2+) stores. Given the recently discovered sensitization of Ca(2+) release channels (ryanodine receptors; RyRs) of the sarcoplasmic reticulum (SR) by luminal SR Ca(2+), waves could also be driven by RyR sensitization, mediated by SR overloading via Ca(2+) pump (SERCA), acting in tandem with CICR. METHODS: Confocal imaging of the Ca(2+) indicator fluo-3 was combined with UV-flash photolysis of caged compounds and the whole-cell configuration of the patch clamp technique to carry out these experiments in isolated guinea pig ventricular cardiomyocytes. RESULTS: Upon sudden slowing of the SERCA in cardiomyocytes with a photoreleased inhibitor, waves indeed decelerated immediately. No secondary changes of Ca(2+) signaling or SR Ca(2+) content due to SERCA inhibition were observed in the short time-frame of these experiments. CONCLUSIONS: Our findings are consistent with Ca(2+) loading resulting in a zone of RyR 'sensitization' traveling within the SR, but inconsistent with CICR as the predominant mechanism driving the Ca(2+) waves. This alternative mode of RyR activation is essential to fully conceptualize cardiac arrhythmias triggered by spontaneous Ca(2+) release.

DAPK2 is a novel E2F1/KLF6 target gene involved in their proapoptotic function

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

Calcium channel TRPV6 is involved in murine maternal-fetal calcium transport

Maternal-fetal calcium (Ca(2+)) transport is crucial for fetal Ca(2+) homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca(2+) channel in maternal-fetal Ca(2+) transport was investigated using Trpv6 knockout mice. The Ca(2+) concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca(2+) ((45)Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal-fetal Ca(2+) transport in mice. These expression sites were co-localized with calbindin D(9K) in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal-fetal Ca(2+) transport. We propose that TRPV6 functions as a Ca(2+) entry pathway, which is critical for fetal Ca(2+) homeostasis.

The Wabar impact craters, Saudi Arabia, revisited

The very young Wabar craters formed by impact of an iron meteorite and are known to the scientific community since 1933. We describe field observations made during a visit to the Wabar impact site, provide analytical data on the material collected, and combine these data with poorly known information discovered during the recovery of the largest meteorites. During our visit in March 2008, only two craters (Philby-B and 11 m) were visible; Philby-A was completely covered by sand. Mapping of the ejecta field showed that the outcrops are strongly changing over time. Combining information from different visitors with our own and satellite images, we estimate that the large seif dunes over the impact site migrate by approximately 1.0–2.0 m yr␣1 southward. Shock lithification took place even at the smallest, 11 m crater, but planar fractures (PFs) and undecorated planar deformation features (PDFs), as well as coesite and stishovite, have only been found in shock-lithified material from the two larger craters. Shock-lithified dune sand material shows perfectly preserved sedimentary structures including cross-bedding and animal burrows as well as postimpact structures such as open fractures perpendicular to the bedding, slickensides, and radiating striation resembling shatter cones. The composition of all impact melt glasses can be explained as mixtures of aeolian sand and iron meteorite. We observed a partial decoupling of Fe and Ni in the black impact glass, probably due to partitioning of Ni into unoxidized metal droplets. The absence of a Ca-enriched component demonstrates that the craters did not penetrate the bedrock below the sand sheet, which has an estimated thickness of 20–30 m.