A point mutation in an unusual Sec7 domain is linked to brefeldin A resistance in a Plasmodium falciparum line generated by drug selection

The malaria parasite Plasmodium falciparum has an unusual organization of its secretory compartments. As an approach to a functional identification of auxiliary proteins involved in secretion, a parasite line was generated by drug selection that is resistant to brefeldin A, an inhibitor of the secretory pathway. In the resistant line, neither protein secretion nor parasite viability were affected by the drug. The analysis of a sec7 domain, a conserved structure of guanine nucleotide exchange factors (ARF-GEF) required for the activation of ADP-ribosylation factors, revealed a single methionine-isoleucine substitution in the resistant parasite line. ARF-GEFs are key molecules in the formation of transport vesicles and the main targets of brefeldin A. The methionine residue in this position of sec7 domains is highly conserved and confers brefeldin A sensitivity. Unlike other eukaryotes that have multiple ARF-GEFs, the plasmodial genome encodes a single sec7 domain. This domain shows a distinct structural difference to all sec7 domains analysed so far; two conserved subdomains that are essential for protein function are separated in the plasmodial protein by an insertion of 146 amino acids.     

Expression of Drosophila Ten-a,a dimeric receptor during embryonic development

Ten-a and Ten-m are the two Drosophila members of the newly discovered Ten-m family of dimeric type II transmembrane proteins. Here, we report complete cDNA cloning and protein expression patterns of Ten-a. The Ten-a protein, a dimeric receptor of about 500 kDa is mainly expressed on axons of the embryonic central nervous system and on muscle attachment sites.     

CaGE: cardiac gene expression knowledgebase

CaGE is a Cardiac Gene Expression knowledgebase we have developed to facilitate the analysis of genes important to human cardiac function. CaGE integrates the functionality of the LocusLink database with data from several human cardiac expression libraries, phenotypic data from OMIM and data from large-scale microarray gene expression studies to create a knowledgebase of gene expression in human cardiac tissue. The knowledgebase is fully searchable via the web using several intuitive query interfaces. Results can be displayed in several concise easy to navigate formats. AVAILABILITY: CaGE is located at http://www.cage.wbmei.jhu.edu     

Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.     

Weight stability masks sarcopenia in elderly men and women

Skeletal muscle loss or sarcopenia in aging has been suggested in cross-sectional studies but has not been shown in elderly subjects using appropriate measurement techniques combined with a longitudinal study design. Longitudinal skeletal muscle mass changes after age 60 yr were investigated in independently living, healthy men (n = 24) and women (n = 54; mean age 73 yr) with a mean +/- SD follow-up time of 4.7 +/- 2.3 yr. Measurements included regional skeletal muscle mass, four additional lean components (fat-free body mass, body cell mass, total body water, and bone mineral), and total body fat. Total appendicular skeletal muscle (TSM) mass decreased in men (-0.8 +/- 1.2 kg, P = 0.002), consisting of leg skeletal muscle (LSM) loss (-0.7 +/- 0.8 kg, P = 0.001) and a trend toward loss of arm skeletal muscle (ASM; -0.2 +/- 0.4 kg, P = 0.06). In women, TSM mass decreased (-0.4 +/- 1.2 kg, P = 0.006) and consisted of LSM loss (-0.3 +/- 0.8 kg, P = 0.005) and a tendency for a loss of ASM (-0.1 +/- 0.6 kg, P = 0.20). Multiple regression modeling indicates greater rates of LSM loss in men. Body weight in men at follow-up did not change significantly (-0.5 +/- 3.0 kg, P = 0.44) and fat mass increased (+1.2 +/- 2.4 kg, P = 0.03). Body weight and fat mass in women were nonsignificantly reduced (-0.8 +/- 3.9 kg, P = 0.15 and -0.8 +/- 3.5 kg, P = 0.12). These observations suggest that sarcopenia is a progressive process, particularly in elderly men, and occurs even in healthy independently living older adults who may not manifest weight loss.     

Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles

Plasmid-encoded GM-CSF (pGM-CSF) is an adjuvant for genetic vaccines; however, little is known about how pGM-CSF enhances immunogenicity. We now report that pGM-CSF injected into mouse muscle leads to a local infiltration of potential APCs. Infiltrates reached maximal size on days 3 to 5 after injection and appeared in several large discrete clusters within the muscle. Immunohistological studies in muscle sections from mice injected with pGM-CSF showed staining of cells with the macrophage markers CD11b, Mac-3, IA(d)/E(d) and to the granulocyte marker GR-1 from day 1 through day 14. Cells staining with the dendritic cell marker CD11c were detected only on days 3 to 5. Muscles injected with control plasmids did not stain for CD11c but did stain for CD11b, Mac-3, IA(d)/E(d), and GR-1. No staining was observed with the APC activation markers, B7.1 or CD40, or with markers for T or B cells. These findings are consistent with the infiltrating cells in the pGM-CSF-injected muscles being a mixture of neutrophils, macrophages, and immature dendritic cells and suggest that the i.m. APCs may be enhancing immune responses to coinjected plasmid Ags. This hypothesis is supported by data showing that 1) separation of injections with pGM-CSF and Ag-expressing plasmid into different sites did not enhance immune responses and 2) immune enhancement was associated with the presence of CD11c+ cells in the infiltrates. Thus, pGM-CSF enhancement may depend on APC recruitment to the i.m. site of injection.     

Multiple isoforms of porcine aromatase are encoded by three distinct genes

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.     

Characterisation of the high-molecular weight fructan isolated from garlic (Allium sativum L.)

A high molecular weight fructan was isolated from garlic and the structure determined by enzymatic, chemical and spectroscopic (NMR) methods. It was found that the garlic fructan belongs to the neokestose family. It has a (2 --> 1)-linked beta-D-Fruf backbone with (2 --> 6)-linked beta-D-Fruf side chains. A structural model was postulated for a degree of polymerisation of about 58. This model was substantiated using an endo-inulinase purified from Aspergillus ficuum and by 1H and 13C NMR spectroscopy.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.