Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: State of the science and planning the way forward

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Lipid raft targeting of hematopoietic protein tyrosine phosphatase by protein kinase C theta-mediated phosphorylation

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.     

The role of microarthropods in terrestrial decomposition: a meta-analysis of 40 years of litterbag studies

Litterbags have been utilized in soil ecology for about 50 years. They are useful because they confine organic material and thus enable the study of decomposition dynamics (mass loss and/or nutrient loss through time, colonization by soil biota) in situ, i.e. under field conditions. Researchers can easily restrict or permit access to certain size classes of soil fauna to determine their contribution to litter mass loss by choosing adequate mesh size or applying specific biocides. In particular, the mesofauna has received much attention since it comprises two very abundant and diverse microarthropod groups, the Collembola (springtails) and Acari (mites). We comprehensively searched the literature from the mid‐1960s to the end of 2005 for reports on litterbag experiments investigating the role of microarthropods in terrestrial decomposition. Thirty papers reporting 101 experiments satisfied our selection criteria and were included in the database. Our meta‐analysis revealed that microarthropods have a moderate but significant effect on mass loss. We discuss in detail the interactions of the microarthropod effect with study characteristics such as experimental design (e.g. number of bags, duration of experiment), type of exposed organic matter, climatic zone and land use of the study site. No publication bias was detected; however, we noticed a significant decrease in the microarthropod effect with publication year, indicating that, in the first decades of litterbag use, soil zoologists may have studied “promising” sites with a higher a priori probability of positive microarthropod effects on litter mass loss. A general weakness is that the treatments differ not only with respect to the presence or absence of microarthropods, but also with regard to mesh size (small to exclude microarthropods, wide to permit their access) or presence (to exclude microarthropods) and absence (to permit their access) of an insecticide. Consequently, the difference between the decomposition rates in the treatments is not a pure microarthropod effect but will be influenced by the additive effects of mesh size and insecticide. The relative contribution of the “true” microarthropod effect remains unknown without additional treatments controlling for the differential mesh size/insecticide effect. A meta‐analysis including only those studies using different mesh size and for which the data were corrected by subtracting an estimated mesh size effect based on data from the literature yielded a significantly negative microarthropod effect on litter decomposition. These results cast doubt on the widely accepted hypothesis that microarthropods generally exert a positive effect on litter mass loss. We conclude that after 40 years of litterbag studies our knowledge on the role of microarthropods in litter mass loss remains limited and that the inclusion of a third treatment in future studies is a promising way to retain litterbags as a meaningful tool of soil biological studies.     

Discovery of matrix metalloproteases selective and activated peptide–doxorubicin prodrugs as anti-tumor agents

To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide–Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox.     

Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.     

A GC cluster repeat is a hotspot for mit- macro-deletions in yeast mitochondrial DNA.

Summary In a random collection of mit^– mutations of the yeast strain 777-3A we find that deletions are exceptionally frequent in the OXI3 gene, a large mosaic gene coding for subunit I of cytochrome oxidase. About 10% of all oxi3 ^– mutants carry the same macro-deletion, del-A, extending from the 5 non-translated leader of OXI3 to intron 5b of this gene. Determination of the respective wild-type sequences and of the del-A junction sequence revealed that the end-points of the deletion are in two GC clusters with 31 by sequence identity which are located at a distance of 11.3 kb. We speculate that not only the sequence identity of the two GC clusters but also the palindromic structure of these putatively mobile elements of yeast mitochondrial DNA (mtDNA) plays a role in deletion formation.     

Self-incompatibility in passionfruit: evidence of gametophytic-sporophytic control

Self-incompatibility in passionfruit was studied in families originated from crosses among plants that presented differences in reciprocal crosses. The three families, obtained by crossing S(3) plants, exhibited one incompatible group; no reciprocal differences were observed. The phenotype of the families was the same as the parent plants, S(3). These results suggest the presence of a gene ( G), gametophytic in its action, associated to the sporophytic gene S, modifying the incompatibility reaction in passionfruit. The reciprocal difference exhibited in the crosses among the parents could be explained as a matching between plants homozygous for S, but homozygous and heterozygous for G. Actually this would be a partially compatible cross, not detectable when the evaluation is done based on fruit set data. As the family originated from this kind of cross is homozygous for S and heterozygous for G, no reciprocal differences are expected, and the phenotype should be the same as the parental plants, as observed in the present work.     

SC1/hevin. An extracellular calcium-modulated protein that binds collagen I

SC1, a member of the BM-40 family of extracellular matrix proteins, was recombinantly expressed in a eukaryotic expression system. The full-length protein as well as truncated versions were purified to homogeneity under non-denaturing conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of full-length SC1 revealed a mass of 87.8 kDa of which 16.8 kDa is contributed by posttranslational modifications. In electron microscopy, after negative staining, SC1 was revealed as a globule attached to a thread-like structure. A calcium dependence of the SC1 conformation could be demonstrated by fluorescence spectroscopy. In the extracellular matrix of cultured osteosarcoma cells SC1 was found associated with collagen I-containing fibrils, and binding of SC1 to reconstituted collagen I fibrils could be demonstrated by immunogold labeling and electron microscopy. SC1 showed a broad expression in a variety of tissues.