A deficiency screen of the major autosomes identifies a gene (matrimony) that is haplo-insufficient for achiasmate segregation in Drosophila oocytes

In Drosophila oocytes, euchromatic homolog-homolog associations are released at the end of pachytene, while heterochromatic pairings persist until metaphase I. A screen of 123 autosomal deficiencies for dominant effects on achiasmate chromosome segregation has identified a single gene that is haplo-insufficient for homologous achiasmate segregation and whose product may be required for the maintenance of such heterochromatic pairings. Of the deficiencies tested, only one exhibited a strong dominant effect on achiasmate segregation, inducing both X and fourth chromosome nondisjunction in FM7/X females. Five overlapping deficiencies showed a similar dominant effect on achiasmate chromosome disjunction and mapped the haplo-insufficient meiotic gene to a small interval within 66C7-12. A P-element insertion mutation in this interval exhibits a similar dominant effect on achiasmate segregation, inducing both high levels of X and fourth chromosome nondisjunction in FM7/X females and high levels of fourth chromosome nondisjunction in X/X females. The insertion site for this P element lies immediately upstream of CG18543, and germline expression of a UAS-CG18543 cDNA construct driven by nanos-GAL4 fully rescues the dominant meiotic defect. We conclude that CG18543 is the haplo-insufficient gene and have renamed this gene matrimony (mtrm). Cytological studies of prometaphase and metaphase I in mtrm hemizygotes demonstrate that achiasmate chromosomes are not properly positioned with respect to their homolog on the meiotic spindle. One possible, albeit speculative, interpretation of these data is that the presence of only a single copy of mtrm disrupts the function of whatever "glue" holds heterochromatically paired homologs together from the end of pachytene until metaphase I.     

Proteasomal activity modulates TGF-ss signaling in a gene-specific manner

Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F-actin)-rich structures. The current work reveals that an amino-terminal segment of MHCK A, previously identified as forming a coiled-coil, mediates anterior localization. MHCK A co-sediments with F-actin, and deletion of the amino-terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F-actin, and reveal the presence of an actin-binding function not previously detected by primary sequence evaluation of the coiled-coil domain.     

Liposome (Lipodine)-mediated DNA vaccination by the oral route

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine liposomes composed of 16 micro moles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 micro moles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 micro moles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1 : 0.5 : 0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 micro g liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 micro g of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine) may be a useful system for the oral delivery of DNA vaccines.     

Interaction of folk medicinal plant extracts with human alpha2-adrenoceptor subtypes

Forty-two extracts of folk medicinal plant organs from Pakistan were tested in competition binding assays for their interaction with the specific ligand recognition sites on the human alpha2-adrenoceptor subtypes alpha2A, alpha2B and alpha2C Strong binding of the extracts (40 mg/ml) from Acacia nilotica (L.) Delile leaves (88-98% displacement of radiolabel) and Peganum harmala seeds (89-96% displacement) on three subtypes prompted us to extract these plant materials with 40% and 80% methanol, ethanol, and acetone. The extraction results indicated an absence of alpha2-adrenoceptor binding activity in the stalk of A. nilotica and A. tortils, whereas the leaves of both plants contained activity. The extracts of A. nilotica leaves showed a slight, but consistent, preference for the alpha2C-adrenoceptor, whereas the leaves of A. tortils were slightly more active on the alpha2B subtype. The extract of P. harmala stalks was less active than that of its seeds. The binding activities of A. nilotica leaves and P. harmala seeds were mainly concentrated in the water and 30% methanol fractions and further sub-fractions. In a functional activity assay, the active fractions inhibited epinephrine-stimulated 35S-GTPyS binding, thus indicating a predominantly antagonistic nature of the compounds with alpha2-adrenoceptor affinity in these fractions. Among the known major alkaloids of P. harmala (demissidine, harmaline, harmine, 6-methoxyharmalan, and norharmane), only 6-methoxyharmalan showed moderate affinity (dissociation constant (Ki) of 530 +/- 40 nm for alpha2A subtype). This study is a first systematic attempt towards the discovery of potential drug candidates from these plant materials for treating alpha2-adrenoceptor related diseases.     

Characterization of hemocytes from the yellow fever mosquito,Aedes aegypti

Mosquitoes are the most important arthropod disease vectors, transmitting a broad range of pathogens that cause diseases such as malaria, lymphatic filariasis, and yellow fever. Mosquitoes and other insects are able to mount powerful cellular and humoral immune responses against invading pathogens. To date, most studies have concentrated on the humoral response. In the current study we describe the hemocytes (blood cells) of the yellow fever mosquito, Aedes aegypti, by means of morphology, lectin binding, and enzyme activity and immunocytochemistry. Our light and electron microscopic studies suggest the presence of four distinct hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. We believe granulocytes and oenocytoids are true circulating hemocytes, but adipohemocytes and thrombocytoids are likely adhered to fixed tissues. Granulocytes, the most abundant cell type, have acid phosphatase and alpha-naphthyl acetate esterase activity, and bind the exogenous lectins WGA, HPA, and GNL. Phenoloxidase, an essential enzyme in the melanotic encapsulation immune response, was detected inside oenocytoids. This is, to our knowledge, the first report that has detected phenoloxidase inside mosquito hemocytes at the ultrastructural level. These results have begun to form a knowledge base for our ongoing studies on the function of Ae. aegypti hemocytes, and their involvement in controlling infections.     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

Genetically modified laboratory animals--what welfare problems do they face?

In this article, we respond to public concern expressed about the welfare of genetically modified (GM) nonhuman animals. As a contribution to the debate on this subject, we attempt in this article to determine in what situations the practice of genetic modification in rodents may generate significant welfare problems. After a brief discussion of the principles of animal welfare, we focus on the problem of animal suffering and review some types of gene modifications likely to cause predictable welfare problems. In this article, we also consider suffering that may be involved in the process of generating GM animals. Finally, we discuss the role of GM animals in attempts to reduce, replace, and refine the use of animals in research.     

Microfilament network is needed for the endocytosis of water channels and not for apical membrane insertion upon vasopressin action

In the current study, a novel role for the microfilaments in vasopressin-induced water transport in toad urinary bladders, a popular model for the mammalian collecting duct, was established. Vasopressin-induced water transport was not affected by cytochalasin D (CD, 20 microM) or latrunculin B (Lat B, 0.5-2 microM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels and insertion of membranes into the apical membrane are microfilament-independent. After the removal of vasopressin, bladders treated with CD or Lat B continued to transport water at least 2-3-fold greater than those that received the vehicle. Furthermore, the enhanced water transport was inhibited by HgCl2 (1 mM), a potent inhibitor of water channel-mediated water flow, suggesting that the enhanced water flow was through water channels. In addition, Lat B and CD inhibited vasopressin-induced endocytosis of horseradish peroxidase (HRP), a fluid endocytotic marker. These results suggested that although microfilaments are not needed for the initial trafficking of water channels to the apical side, the microfilament network is essential for the retrieval of water channels following their insertion into apical membranes.