Mechanism of transport of vegetative storage proteins to the vacuole of the paraveinal mesophyll of soybean leaf

Summary Microscopy techniques were used to identify the pathway of transport of soybean leaf vegetative storage proteins (VSP/ and VSP94) to the vacuoles of a specialized cell type, the paraveinal mesophyll (PVM), where they accumulate. PVM cells are enriched in endoplasmic reticulum and Golgi bodies relative to surrounding mesophyll cells. The margins of medial and trans Golgi cisternae had attached or closely associated noncoated vesicles with densely staining membranes and lumenal contents of the same appearance as material that accumulated in the vacuole. These vesicles appeared to be transported preferentially to the tonoplast, where fusion with the membrane released the granular contents into the vacuole. Cytochemical staining with phosphotungstic acid and silver methenamine supported this interpretation as both the Golgi vesicles and the tonoplast stained intensely with these reagents, unlike the tonoplast of mesophyll cells which do not accumulate VSP. Immunocytochemical localization for VSP/ labeled the Golgi bodies and associated vesicles, and vacuolar material in PVM cells, but not in mesophyll. Similar labeling was seen in PVM of another legume species previously found to accumulate antigenically similar VSPs. Immunolocalization for VSP94, a lipoxygenase, labeled the PVM cytosol and material in the PVM vacuole, but not the Golgi or vesicles. The results of this study demonstrate that the Golgi pathway is utilized for transport of VSP/ in the PVM, which follows the mechanism of deposition demonstrated for certain seed storage proteins. VSP94 appeared to follow a separate path for accumulation in PVM vacuoles.Abbreviations LOX lipoxygenase - PVM paraveinal mesophyll - RER rough endoplasmic reticulum - TEM transmission electron     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Pathway of Phloem unloading of sucrose in corn roots

The pathway of phloem unloading and the metabolism of translocated sucrose were determined in corn (Zea mays) seedling roots. Several lines of evidence show that exogenous sucrose, unlike translocated sucrose, is hydrolyzed in the apoplast prior to uptake into the root cortical cells. These include (a) presence of cell wall invertase activity which represents 20% of the total tissue activity; (b) similarity in uptake and metabolism of [¹⁴C]sucrose and [¹⁴C]hexoses; and (c) randomization of ¹⁴C within the hexose moieties of intracellular sucrose following accumulation of [¹⁴C] (fructosyl)sucrose. Conversely, translocated sucrose does not undergo apoplastic hydrolysis during unloading. Asymmetrically labeled sucrose ([¹⁴C](fructose)sucrose), translocated from the germinating kernels to the root, remained intact indicating a symplastic pathway for unloading. In addition, isolated root protoplasts and vacuoles were used to demonstrate that soluble invertase activity (V_max = 29 micromoles per milligram protein per hour, K_m = 4 millimolar) was located mainly in the vacuole, suggesting that translocated sucrose entered via the symplasm and was hydrolyzed at the vacuole prior to metabolism.     

A population at high risk for esophageal cancer in the north-east of Italy

Esophageal cancer is generally characterised by relatively low incidence and mortality rates in Europe. However, a high-risk population for this tumour is resident in the north-east of Italy. Several studies have been conducted on this population of males confirming the major role of alcohol and tobacco consumption alone and in combination. The inhabitants of this area of Italy constitute an ideal target population for studies of molecular epidemiology aimed at elucidating the natural history of the disease which is still ill-defined, and the distribution of genetic alterations at a population level.     

Design-based mapping of tree attributes by 3P sampling

The estimation of individual values (marks) in a finite population of units (e.g., trees) scattered onto a survey region is considered under 3P sampling. For each unit, the mark is estimated by means of an inverse distance weighting interpolator. Conditions ensuring the design-based consistency of maps are considered under 3P sampling. A computationally simple mean squared error estimator is adopted. Because 3P sampling involves the prediction of marks for each unit in the population, prediction errors rather than marks can be interpolated. Then, marks are estimated by the predictions plus the interpolated errors. If predictions are good, prediction errors are more smoothed than raw marks so that the procedure is likely to better meet consistency requirements. The purpose of this paper is to provide theoretical and empirical evidence on the effectiveness of the interpolation based on prediction errors to prove that the proposed strategy is a tool of general validity for mapping forest stands.     

Ca2+-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins

Despite recent progress in the molecular characterization of high-conductance Ca²⁺-activated K⁺ (maxi-K) channels, the molecular identities of intermediate conductance Ca²⁺-activated K⁺ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca²⁺-activated K⁺ channels (Gardos pathway) of human and rabbit red cells. With studies on K⁺ transport and on binding of ¹²⁵I-charybdotoxin (ChTX) and ¹²⁵I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca²⁺-activated K⁺ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1–37 and 1–38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound ¹²⁵I-charybdotoxin, a novel property for K⁺ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.We thank Dr. Chris Miller of Brandeis University for generously providing recombinant ChTX mutants, Dr. Maria Garcia of Merck Research Laboratories for MgTX and Dr. Regine Romi of Laboratoire d'Ingenierie des Proteines (Marseille, France) for synthetic KTX,1–37 and KTX,1–38. This research was supported by grant HL-15157 from the National Institutes of Health.     

Effect of vanadate of PHA-induced proliferation of human lymphocytes from young and old subjects

The effect of sodium orthovanadate on mitogen-induced proliferation of lymphocytes from young and old human subjects is reported. We found that vanadate is not mitogenic per se; it has an inhibitory effect during the first 3 days of culture, when both differentiation and proliferation take place; it enhances DNA synthesis, acting as a co-mitogen, in the following days of culture, when proliferation prevails. In spite of the fact that lymphocytes from the two groups differ in their responsiveness to PHA and in the activity of (Na+,K+)ATPase, no difference was found as for the effects of vanadate.     

Telomere length in fibroblasts and blood cells from healthy centenarians

Several lines of evidence indicate that telomere shortening during in vitro aging of human somatic cells plays a causal role in cellular senescence. A critical telomere length seems to be associated with the replicative block characterizing senescent cells. In this paper we analyzed the mean length of the terminal restriction fragments (TRF) in fibroblast strains from 4 healthy centenarians, that is, in cells aged in vivo, and from 11 individuals of different ages. No correlation between mean TRF length and donor age was found. As expected, telomere shortening was detected during in vitro propagation of centenarian fibroblasts, suggesting that in fibroblasts aged in vivo telomeres can be far from reaching a critical length. Accordingly, chromosome analysis did not show the presence of telomeric associations in early passage centenarian fibroblasts. In blood cells from various individuals, the expected inverse correlation between mean TRF length and donor age was found. In particular, a substantial difference (about 2 kb) between telomere length in the two cell types was observed in the same centenarian. Expression analysis of three senescence-induced genes, i.e., fibronectin, apolipoprotein J, and p21, revealed for only the fibronectin expression levels a clear positive correlation with donor age. Our results suggest that (1) telomere shortening could play a different role in the aging of different cell types and (2) the characteristics of fibroblasts aged in vitro might not be representative of what occurs in vivo.     

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.