Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

Light and temperature regulation of greening in dark‐grown ginkgo (Ginkgo biloba)

The last steps of chlorophyll (Chl) biosynthesis were studied at different light intensities and temperatures in dark‐germinated ginkgo (Ginkgo biloba L.) seedlings. Pigment contents and 77 K fluorescence emission spectra were measured and the plastid ultrastructure was analysed. All dark‐grown organs contained protochlorophyllide (Pchlide) forms with similar spectral properties to those of dark‐grown angiosperm seedlings, but the ratios of these forms to each other were different. The short‐wavelength, monomeric Pchlide forms were always dominating. Etioplasts with small prolamellar bodies (PLBs) and few prothylakoids (PTs) differentiated in the dark‐grown stems. Upon illumination with high light intensities (800 μmol m^?2 s^?1 photon flux density, PFD), photo‐oxidation and bleaching occurred in the stems and the presence of ¹O₂ was detected. When Chl accumulated in plants illuminated with 15 μmol m^?2 s^?1 PFD it was significantly slower at 10°C than at 20°C. At room temperature, the transformation of etioplasts into young chloroplasts was observed at low light, while it was delayed at 10°C. Grana did not appear in the plastids even after 48 h of greening at 20°C. Reaccumulation of Pchlide forms and re‐formation of PLBs occurred when etiolated samples were illuminated with 200 μmol m^?2 s^?1 PFD at room temperature for 24 h and were then re‐etiolated for 5 days. The Pchlide forms appeared during re‐etiolation had similar spectral properties to those of etiolated seedlings. These results show that ginkgo seedlings are very sensitive to temperature and light conditions during their greening, a fact that should be considered for ginkgo cultivation.     

Neonatal handling increases sensitivity to acute neurodegeneration in adult rats

Environmental stimuli during the perinatal period can result in persistent individual differences in neural viability and cognitive functions. Earlier studies have shown that brief daily maternal separation and/or handling of rat pups during the first weeks of life reduces stress reactivity during adulthood and attenuates neuronal loss and cognitive decline during aging. In the present study we examined whether neonatal handling also affects the sensitivity of the adult brain to an acute neurotoxic insult. Postnatally handled and nonhandled control rats were left undisturbed from weaning onwards until the age of 11 months. At this age, the animals were subjected to a neurotoxic challenge by unilateral infusion of 60 mM of the glutamate analogue N-methyl-D-aspartate (NMDA) into the nucleus basalis magnocellularis (NBM). The brains were collected to measure cholinergic cell and fiber loss. In the nonlesioned side of the brain, cholinergic cell number in the NBM and fiber density in the cortex were not different between postnatally handled and control rats. However, in the lesioned hemisphere handled animals exhibited a significantly higher loss of choline-acetyltransferase-immunoreactive and acetylcholinesterase-positive fibers in the somatosensory cortex. The present results provide evidence for an enhanced vulnerability of postnatally handled rats to acute neurodegeneration in contrast to the previously reported attenuation of spontaneous aging-related neurodegenerative processes.     

Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome–human serum albumin association

Fluorescence correlation spectroscopy was used to measure the diffusion behavior of a mixture of DMPC or DMPC/DMPG liposomes with human serum albumin (HSA) and mesoporphyrin (MP), which was used as the fluorescent label for liposomes and HSA as well. For decomposing the fluorescence intensity autocorrelation function (ACF) into components corresponding to a liposome population, HSA and MP, we used a maximum entropy procedure that computes a distribution of diffusion times consistent with the ACF data. We found that a simple parametric non-linear fit with a discrete set of decay components did not converge to a stable parameter set. The distribution calculated with the maximum entropy method was stable and the average size of the particles calculated from the effective diffusion time was in good agreement with the data determined using the discrete-component fit.     

Serglycin constitutively secreted by myeloma plasma cells is a potent inhibitor of bone mineralization in vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n=2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.     

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.     

Distribution of the Eastern knapweed fritillary (Melitaea ornata Christoph, 1893) (Lepidoptera: Nymphalidae): past, present and future

Climatic change during the Quaternary resulted in periodical range restrictions and expansions in most temperate species. Although some repetitive patterns have been supported, it became obvious that species’ responses might be rather specific and may also depend on habitat preferences of the species in question. Distribution of Melitaea ornata, a little known fritillary species is analysed on different time scales using MaxEnt software. Using the results of genitalia morphometry and the predicted potential refugia during the Last Glacial Maximum (LGM), we reconstructed probable re-colonisation routes. We also predicted changes in the potential area for 2080. The present distribution fits well the known occurrence data except for the Iberian Peninsula and North-Africa where the species is missing. Based on our predictions, temperate areas seem to be less suitable for the species. We proposed two hypotheses to explain this pattern: a less probable recent extinction from Iberia and a more supported historical explanation. Predicted distribution during the LGM mainly fits to widely accepted refugia. Europe was probably re-colonised from two main sources, from the Apennine peninsula and from the Balkans which was probably connected to the Anatolian refugia. Populations of the Levant region and in the Elburs Mts. do not show any significant expansion. Further studies are necessary in the case of the predicted Central Asian refugia. Predictions for 2080 show a northward shift and some extinction events in the Mediterranean region. Core areas are identified which might have a potential for expansion including southern Russia, Hungary and possibly Provence in France. Predicted northward area shifts are only possible if the potential leading edge populations and habitats of the species can be preserved.     

Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.     

An antibody specific for coagulation factor IX enhances the activity of the intrinsic factor X-activating complex

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (kcat) of the enzyme complex increased 2- to 3-fold whereas the Km for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.