Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.     

Karyological studies of some representatives of <Emphasis Type="Italic">Tanacetum</Emphasis> L. (Anthemideae-Asteraceae) from north-east Anatolia

The karyotypes of nine Tanacetum taxa distributed in north-east Anatolia, Turkey, were determined and evaluated by cluster analysis and principal-components analysis. Chromosome numbers were 2n = 2x = 18 (8 taxa) and 4x = 36 (1 taxon). Somatic chromosome numbers of two taxa and a new ploidy level in one taxon are reported for the first time. Karyotype analysis indicated that chromosomes of Tanacetum taxa have predominantly median centromeres. The taxa studied differed significantly in the size of the short arms and long arms, and the arm ratio of each pair of homologous chromosomes, indicating structural rearrangements of the chromosomes have been involved in diversification of the taxa. They were placed in 2A, 3A, and 2B of Stebbins’ karyotype classification, showing the presence of a primitive symmetrical karyotype in the genus. Several systematic and evolutionary aspects of the genus are discussed on the basis of karyological data.     

Diterpenoid alkaloids of Aconitum vulparia Rchb

From the roots of Aconitum vulparia Rchb., collected in Prüm (Germany), a new norditerpenoid alkaloid, named alexhumboldtine, has been isolated along with the known norditerpenoid alkaloids lappaconitine, anthranoyllycoctonine, lycoctonine, puberaconitine, ajacine, and septentriodine. The structure of alexhumboldtine was established on the basis of 1H, 13C, DEPT, homonuclear 1H COSY, NOESY, HSQC, and HMBC NMR studies. From the aerial parts of the plant another norditerpenoid alkaloid, aconorine, has been isolated.     

Hyberbaric oxygen increases atresia in normal &; steroid induced PCO rat ovaries

Background  

In this study, we investigated the effect of hyperbaric oxygen therapy (HBOT) on the morphology of estradiol valerate (EV) induced polycystic ovary (PCO) to find a new treatment modality for improvement of PCO.     

Wing size and shape variation of Phlebotomus papatasi (Diptera: Psychodidae) populations from the south and north slopes of the Atlas Mountains in Morocco

The wing shape and size morphology of populations of the medically important phlebotomine sand fly, Phlebotomus papatasi, were examined in two endemic (south of the Atlas Mountains) and nonendemic (north of the Atlas Mountains) foci of cutaneous leishmaniasis by using geometric morphometrics in Morocco. Although it is present in all of Morocco, P. papatasi is the main vector of Leishmania major in only southern part of the Atlas Mountains. There are four major mountain ranges that serve as geographical barriers for species distribution in the study area and at least four gaps were recognized among these barriers. We found statistically significant differences in wing shape morphology between southern and northern populations. Analysis clearly recognized two main groups of populations on both sides of the mountains. The graphical depiction of Principal Component Analysis (PCA) and Canonical Variates Analysis (CVA) confirmed our morphometric study suggesting that the difference in wing morphology between the populations indicates that the population of P. papatasi shows phenotypic plasticity in the study area. According to centroid size analyses, which were used as measures of wing size differences among different sites, the north population of P. papatasi had relatively larger wings than the south population.     

Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons

Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2-based ratiometric Ca(2+)-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 μM, 100 μM and 300 μM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca(2+) imaging protocols revealed that HiK(+) (30 mM)-induced intracellular free Ca(2+) ([Ca(2+)](i)) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 μM, 100 μM and 300 μM LEV (respectively), without any significant effect on basal levels of [Ca(2+)](i). This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca(2+), providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.     

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.     

Synergie between molecular imprinted polymer based on solid-phase extraction and quartz crystal microbalance technique for 8-OHdG sensing

Recently, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been used as a marker to determine the oxidative stress. There is no any cheap and easy determination method based on chips and sensor systems for the determination of 8-OHdG. In this study, we have proposed imprinting methods for 8-OHdG recognition and determination using methacryloylamidohistidine-platinum(II) [MAH-Pt(II)] as a new metal-chelating monomer. The study includes the solid-phase extraction (SPE) of blood sample by a new 8-OHdG imprinted sorbent and the measurement of binding interaction of 8-OHdG imprinted quartz crystal microbalance (QCM) sensor via ligand interaction. 8-OHdG imprinted sorbent has prepared by bulk polymerization of MAH-Pt(II) and N-N'-methylenebisacrylamide. 8-OHdG imprinted sensor has prepared on a QCM chip coating the thiol pretreated Au electrode. At the end of these steps, a thin molecular imprinted polymer (MIP) film for the detection of 8-OHdG has developed and analytical performance of QCM sensor which has prepared using MIP was investigated. The affinity constant (K(a)) for 8-OHdG using MAH-Pt-based thin film has determined by using the Scatchard method. The average percentage recovery of 8-OHdG from plasma samples was found as 80% by using of 8-OHdG imprinted SPE material. At the last step, 8-OHdG level in several blood plasma has been determined by this improved QCM sensor. The obtained results confirmed that the 8-OHdG level in cancer patient's blood was significantly higher than in general subjects.     

Restoration of CA2+-inhibited oxidative phosphorylation in cardiac mitochondria by mitochondrial Ca2+ unloading

Mitochondria, the major source of cellular ATP, display high vulnerability to metabolic stress, in particular to excessive Ca²⁺ loading. Here, we show that Ca²⁺-inhibited mitochondrial ATP generation could be restored through stimulated Ca²⁺ discharge from mitochondrial matrix. This was demonstrated using a Ca²⁺ ionophore or through Na⁺/Ca²⁺ exchange-mediated decrease of mitochondrial Ca²⁺ load. Furthermore, diazoxide, a mitochondrial potassium channel opener, which maintained mitochondrial Ca²⁺ homeostasis, also restored Ca²⁺-inhibited ATP synthesis and preserved the structural integrity of Ca²⁺-challenged mitochondria. Thus, under conditions of excessive mitochondrial Ca²⁺ overload targeting mitochondrial Ca²⁺ transport pathways restores oxidative phosphorylation required for vital cellular processes. This study, therefore, identifies an effective strategy capable to rescue Ca²⁺-disrupted mitochondrial energetics.