Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.     

Loss of keratin 10 is accompanied by increased sebocyte proliferation and differentiation

Here, we present strong evidence that the targeted deletion of keratin 10 (K10) alters sebocyte differentiation in mice, mediated by an increased proliferation and differentiation of cells located in the periphery of the glands. This was not accompanied by the induction of the proliferation-associated keratins K6, K16 and K17. Sebaceous gland cells of K10-/- mice showed an accelerated turnover and secreted more sebum including wax esters, triglycerides, and cholesterol esters. The levels of the major epidermal lipids ceramides and cholesterol were also increased, whereas glycosylceramides and sphingomyelin were decreased which was not based on altered sphingolipid biosynthesis. The amount of Cer(OS), covalently bound to the cornified envelope, remained unchanged, as well as the amount of loricrin and involucrin. In agreement with the unaltered expression of beta-catenin and its targets cyclin D1 and c-Myc, we conclude that the altered composition of the suprabasal intermediate filament cytoskeleton in K10-/- mice increased the differentiation of epidermal stem cells towards the sebocyte lineage.     

Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex

A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as gamma-Tm), but not from either the alpha- or beta-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., (1999). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.     

Calpain-PKC Inter-Relations in Mouse Hippocampus: A Biochemical Approach

In previous studies, we isolated and identified a -calpain/PKC complex from rabbit skeletal muscle. Here, we have used specific purification procedures in order to study the interactions between -calpain and PKC in mouse hippocampus, a brain structure implicated in memory processes. We observed that -calpain and conventional PKCs (, II and ) are co-eluted after anion exchange chromatography. In contrast to our previous results obtained on skeletal muscle, -calpain and PKC isoenzymes were dissociated after gel filtration chromatography. Furthermore, -calpain induced the proteolytic conversion of PKC, II, and into PKM, II, and with a preferential hydrolysis of PKC, a specific isoenzyme of the nervous system. Although the -calpain/PKC interactions in the hippocampus are quite different from skeletal muscle, our results however, point out the functional importance of these inter-relations. Moreover, as PKC has been involved in the biochemical events underlying learning and memory, the preferential relationship between -calpain and PKC promotes the importance of the role that -calpain could play in the cellular mechanisms of memory formation.     

Five Alternative Myosin Converter Domains Influence Muscle Power,Stretch Activation,and Kinetics

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a–e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b–e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.     

TOM70 Sustains Cell Bioenergetics by Promoting IP3R3-Mediated ER to Mitochondria Ca2+ Transfer

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Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in <Emphasis Type="Italic">Vitis cinerea</Emphasis> for marker-assisted selection

Background

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes.

Results

Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F₁ V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM.

Conclusion

The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.