Novel actin-related proteins Arp-T1 and Arp-T2 as components of the cytoskeletal calyx of the mammalian sperm head

The calyx is a large cytoskeletal component of the perinuclear theca of the mammalian sperm head, displaying remarkable morphological interspecies differences, which is biochemically characterized by resistance to high ionic strength and detergents and by a special protein composition, including the basic proteins calicin, cylicin I and II, and two major actin-capping proteins. In our calyx preparations from bull spermatozoa we have noted two major acidic components which upon partial amino acid sequencing have been identified as novel members of the subfamily of actin-related proteins (Arps). Antibodies raised against the corresponding human proteins, termed Arp-T1 and Arp-T2, have been used to detect the proteins by immunoblotting and immunofluorescence microscopy, demonstrating their specific synthesis in the testis, late in spermatid differentiation, and their localization in the calyx. The discovery of two novel Arps as major components in a cytoskeletal, nonmotile structure of mammalian spermatozoa suggests that certain members of this family of proteins may serve functions other than nucleation of actin filaments, and possible biological roles of such Arps in spermatozoa are discussed.     

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

A rapid precolumn high-performance liquid chromatography method based on fluorescence detection has been developed for the measurement of multiple amino acids from both ex vivo and in vivo biological samples using monolithic C18 columns. A mixture of 18 primary amino acids were derivatised with napthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The resulting isoindole derivatives were resolved within 10 min using a linear binary gradient elution profile with Rs values in the range 1.2-9.0. The limit of detection (LOD) was found to be between 6.0 and 60 fmol for 5 microl injection with a signal to noise ratio of 3:1. The NDA derivatives were found to be stable for 9 h at 4 degrees C. This assay has been employed for the rapid analysis of amino acids from brain tissue and microdialysis samples. Examples of application of the method are given.     

Normal somatic hypermutation of Ig genes in the absence of 8-hydroxyguanine-DNA glycosylase

The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and kappa L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1(-/-) and Ogg1(+/+) clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1(-/-) clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.     

Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis

The pleiotropic functions of small GTPase Rho present a challenge to its genetic analysis in multicellular organisms. We report here the use of the MARCM (mosaic analysis with a repressible cell marker) system to analyze the function of RhoA in the developing Drosophila brain. Clones of cells homozygous for null RhoA mutations were specifically labeled in the mushroom body (MB) neurons of mosaic brains. We found that RhoA is required for neuroblast (Nb) proliferation but not for neuronal survival. Surprisingly, RhoA is not required for MB neurons to establish normal axon projections. However, neurons lacking RhoA overextend their dendrites, and expression of activated RhoA causes a reduction of dendritic complexity. Thus, RhoA is an important regulator of dendritic morphogenesis, while distinct mechanisms are used for axonal morphogenesis.     

Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Antigen transmission by replicating antigen-bearing dendritic cells

During steady-state conditions, conventional spleen dendritic cells (DC) turn over every 2-3 days. Recent evidence indicates that in situ proliferation of DC arising from immediate conventional DC precursors is an important contributor to their homeostasis. In this study, we report that replication-competent conventional DC precursors and DC can internalize and transfer model particulate and soluble Ags directly to their DC progeny during cell division. Real-time confocal microscopy and flow cytometry indicated that Ag transmission to progeny was symmetrical, and suggested that other mechanisms of inter-DC Ag transfer were not involved. Soluble protein Ags inherited by DC progeny were presented effectively to Ag-specific T lymphocytes. Furthermore, we show that the number of DC, and the proportion that are actively proliferating, expands several-fold during an immune response against a viral infection. Our results point to an unanticipated mechanism in which DC are continuously replaced from Ag-bearing replication-competent precursor cells that pass Ag molecules onto their progeny through successive cell divisions. Our findings help explain how Ag may persist in a population of DC despite the brief lifespan of individual mature DC.     

Beta-edge interactions in a pentadecameric human antibody V kappa domain

Antibodies are the archetypal molecules of the Ig-fold superfamily. Their highly conserved beta-sheet architecture has evolved to avoid aggregation by protecting edge strands. However, the crystal structure of a human V kappa domain described here, reveals an exposed beta-edge strand which mediates assembly of a helical pentadecameric oligomer. This edge strand is highly conserved in V kappa domains but is both shortened and capped by the use of two sequential trans-proline residues in V lambda domains. We suggest that the exposure of this beta-edge in V kappa domains may explain why light-chain deposition disease is mediated predominantly by kappa antibodies.     

Anopheles gambiae miRNAs as actors of defence reaction against Plasmodium invasion

The path Plasmodium takes across the Anopheles midgut constitutes the major bottleneck during the malaria transmission cycle. In the present study, using a combination of shot-gun cloning and bioinformatic analysis, we have identified 18 miRNAs from Anopheles gambiae including three miRNAs unique to mosquito. Twelve of them are expressed ubiquitously across the body, independently of gender, while the other six exhibited an expression pattern restricted to the digestive system. Strikingly, the expression patterns of four miRNAs, including the three unique to mosquito, are affected by the presence of Plasmodium. We also show that knocking down Dicer1 and Ago1 mRNAs led to an increased sensitivity to Plasmodium infection. Altogether, these data support an involvement of miRNAs as new layers in the regulation of Anopheles defence reaction.