Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly Lucilia cuprina feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar L. cuprina larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of L. cuprina larvae that may have general application to the control of other insect pests.

Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis

SMAD2 is a member of the transforming growth factor β and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior–posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis.

Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene

Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles. It is encoded by a new centrin gene, which we propose to name HsCEN3 (Homo sapiens centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (Mus musculus centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.

Histone underacetylation is an ancient component of mammalian X chromosome inactivation

Underacetylation of histone H4 is thought to be involved in the molecular mechanism of mammalian X chromosome inactivation, which is an important model system for large-scale genetic control in eukaryotes. However, it has not been established whether histone underacetylation plays a critical role in the multistep inactivation pathway. Here we demonstrate differential histone H4 acetylation between the X chromosomes of a female marsupial, Macropus eugenii. Histone underacetylation is the only molecular aspect of X inactivation known to be shared by marsupial and eutherian mammals. Its strong evolutionary conservation implies that, unlike DNA methylation, histone underacetylation was a feature of dosage compensation in a common mammalian ancestor, and is therefore likely to play a central role in X chromosome inactivation in all mammals.

Gravity and the orientation of cell division

A novel culture system for mammalian cells was used to investigate division orientations in populations of Chinese hamster ovary cells and the influence of gravity on the positioning of division axes. The cells were tethered to adhesive sites, smaller in diameter than a newborn cell, distributed over a nonadhesive substrate positioned vertically. The cells grew and divided while attached to the sites, and the angles and directions of elongation during anaphase, projected in the vertical plane, were found to be random with respect to gravity. However, consecutive divisions of individual cells were generally along the same axis or at 90° to the previous division, with equal probability. Thus, successive divisions were restricted to orthogonal planes, but the choice of plane appeared to be random, unlike the ordered sequence of cleavage orientations seen during early embryo development.

Heme oxygenase 1 is required for mammalian iron reutilization

The majority of iron for essential mammalian biological activities such as erythropoiesis is thought to be reutilized from cellular hemoproteins. Here, we generated mice lacking functional heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron, to assess its participation in iron homeostasis. Hmox1-deficient adult mice developed an anemia associated with abnormally low serum iron levels, yet accumulated hepatic and renal iron that contributed to macromolecular oxidative damage, tissue injury, and chronic inflammation. Our results indicate that Hmox1 has an important recycling role by facilitating the release of iron from hepatic and renal cells, and describe a mouse model of human iron metabolic disorders.

Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are ≈65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells

The nematode Caenorhabditis elegans exhibits behavioral responses to many volatile odorants. Chemotaxis toward one such odorant, diacetyl (butanedione), requires the function of a seven-transmembrane receptor protein encoded by the odr-10 gene. To determine directly whether ODR-10 protein is an odorant receptor, it is necessary to express the protein in a heterologous system and show that it responds to diacetyl by activation of a G protein signaling pathway. Here we demonstrate that human cells expressing ODR-10 on their surfaces exhibit a transient elevation in intracellular Ca2+ levels after diacetyl application. Volatile compounds that differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone) are not ODR-10 agonists. Behavioral responses to these compounds are not dependent on odr-10 function, so ODR-10 specificity in human cells resembles in vivo specificity. The apparent affinity of ODR-10 for diacetyl observed in human cells is consistent with the diacetyl concentration ranges that allow efficient nematode chemotaxis. ODR-10 expressed in human cells also responds to two anionic compounds, pyruvate and citrate, which are metabolic precursors used for diacetyl production by certain bacterial species. Ca2+ elevation in response to ODR-10 activation is due to release from intracellular stores.

Structural protein 4.1 is located in mammalian centrosomes

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli. Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a β-lyase activity that nicks DNA 3′ to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7,8-dihydroxy-8-oxo-2′-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix–hairpin–helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

Morphological analysis of the mammalian postcranium: A developmental perspective

The past two decades have greatly improved our knowledge of vertebrate skeletal morphogenesis. It is now clear that bony morphology lacks individual descriptive specification and instead results from an interplay between positional information assigned during early limb bud deployment and its “execution” by highly conserved cellular response programs of derived connective tissue cells (e.g., chondroblasts and osteoblasts). Selection must therefore act on positional information and its apportionment, rather than on more individuated aspects of presumptive adult morphology. We suggest a trait classification system that can help integrate these findings in both functional and phylogenetic examinations of fossil mammals and provide examples from the human fossil record.

Developmental regulation of spine motility in the mammalian central nervous system

The function of dendritic spines, postsynaptic sites of excitatory input in the mammalian central nervous system (CNS), is still not well understood. Although changes in spine morphology may mediate synaptic plasticity, the extent of basal spine motility and its regulation and function remains controversial. We investigated spine motility in three principal neurons of the mouse CNS: cerebellar Purkinje cells, and cortical and hippocampal pyramidal neurons. Motility was assayed with time-lapse imaging by using two-photon microscopy of green fluorescent protein-labeled neurons in acute and cultured slices. In all three cell types, dendritic protrusions (filopodia and spines) were highly dynamic, exhibiting a diversity of morphological rearrangements over short (<1-min) time courses. The incidence of spine motility declined during postnatal maturation, but dynamic changes were still apparent in many spines in late-postnatal neurons. Although blockade or induction of neuronal activity did not affect spine motility, disruption of actin polymerization did. We hypothesize that this basal motility of dendritic protrusions is intrinsic to the neuron and underlies the heightened plasticity found in developing CNS.

Repair of thalassemic human β-globin mRNA in mammalian cells by antisense oligonucleotides

In one form of β-thalassemia, a genetic blood disorder, a mutation in intron 2 of the β-globin gene (IVS2-654) causes aberrant splicing of β-globin pre-mRNA and, consequently, β-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3

YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features. It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes. A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 has been identified. A Gal4 DNA binding domain–mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites. Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1. Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1. These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human.

Regulation of transforming growth factor β- and activin-induced transcription by mammalian Mad proteins

Members of the transforming growth factor β (TGF-β) superfamily are involved in diverse physiological activities including development, tissue repair, hormone regulation, bone formation, cell growth, and differentiation. At the cellular level, these functions are initiated by the interaction of ligands with specific transmembrane receptors with intrinsic serine/threonine kinase activity. The signaling pathway that links receptor activation to the transcriptional regulation of the target genes is largely unknown. Recent work in Drosophila and Xenopus signaling suggested that Mad (Mothers against dpp) functions downstream of the receptors of the TGF-β family. Mammalian Mad1 has been reported to respond to bone morphogenetic protein (BMP), but not to TGF-β or activin. We report here the cloning and functional studies of a novel mammalian Mad molecule, Mad3, as well as a rat Mad1 homologue. Overexpression of Mad3 in a variety of cells stimulated basal transcriptional activity of the TGF-β/activin-responsive reporter construct, p3TP-Lux. Furthermore, expression of Mad3 could potentiate the TGF-β- and activin-induced transcriptional stimulation of p3TP-Lux. By contrast, overexpression of Mad1 inhibited the basal as well as the TGF-β/activin induced p3TP-Lux activity. These findings, therefore, support the hypothesis that Mad3 may serve as a mediator linking TGF-β/activin receptors to transcriptional regulation.