Getting knotted: a model for the structure and activation of Spätzle.

Sequence analyses show that Spätzle, the Drosophila melanogaster Toll-receptor ligand, shows striking similarity to nerve growth factor and coagulogen. Comparative modelling suggests that Spätzle adopts a cystine-knot fold and forms a dimer that contains a single, intermolecular disulphide bridge. Proteolytically cleaved Spätzle could therefore dimerize and activate the Toll receptor by inducing receptor dimerization.

Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.

A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.

Modelling the force of infection for hepatitis B and hepatitis C in injecting drug users in England and Wales.

BACKGROUND: Injecting drug use is a key risk factor, for several infections of public health importance, especially hepatitis B (HBV) and hepatitis C (HCV). In England and Wales, where less than 1% of the population are likely to be injecting drug users (IDUs), approximately 38% of laboratory reports of HBV, and 95% of HCV reports are attributed to injecting drug use. METHODS: Voluntary unlinked anonymous surveys have been performed on IDUs in contact with specialist agencies throughout England and Wales. Since 1990 more than 20,000 saliva samples from current IDUs have been tested for markers of infection for HBV, HCV testing has been included since 1998. The analysis here considers those IDUs tested for HBV and HCV (n = 5,682) from 1998-2003. This study derives maximum likelihood estimates of the force of infection (the rate at which susceptible IDUs acquire infection) for HBV and HCV in the IDU population and their trends over time and injecting career length. The presence of individual heterogeneity of risk behaviour and background HBV prevalence due to routes of transmission other than injecting are also considered. RESULTS: For both HBV and HCV, IDUs are at greatest risk from infection in their first year of injecting (Forces of infection in new initiates 1999-2003: HBV = 0.1076 95% C.I: 0.0840-0.1327 HCV = 0.1608 95% C.I: 0.1314-0.1942) compared to experienced IDUs (Force of infection in experienced IDUs 1999-2003: HBV = 0.0353 95% C.I: 0.0198-0.0596, HCV = 0.0526 95% C.I: 0.0310-0.0863) although independently of this there is evidence of heterogeneity of risk behaviour with a small number of IDUs at increased risk of infection. No trends in the FOI over time were detected. There was only limited evidence of background HBV infection due to factors other than injecting. CONCLUSION: The models highlight the need to increase interventions that target new initiates to injecting to reduce the transmission of blood-borne viruses. Although from the evidence here, identification of those individuals that engage in heightened at-risk behaviour may also help in planning effective interventions. The data and methods described here may provide a baseline for monitoring the success of public health interventions.

Spatial and temporal distribution of a Drosophila melanogaster embryonic variable nuclear antigen.

A polyclonal antibody has been prepared that specifically recognises a nuclear protein antigen in Drosophila embryos. During development, the antigen appears initially to be uniformly distributed but by nuclear division cycle 10 is seen to accumulate in nuclei in a manner suggesting that it is destroyed or becomes modified upon transition from S- to M phase of the nuclear division cycle. This conclusion is supported by the observed disappearance of the antigen from the postblastoderm nuclei in a manner that reflects the pattern of the first asynchronous postblastoderm cell division and persistence in the polyploid nuclei of the amnioserosa that do not undergo further cell or nuclear division. In Western blot experiments, the antibody detects specifically a 105 kDa nuclear protein that probably corresponds to the antigen detected in embryos by immunocytochemical means.

Regulation of translation and proteolysis during the development of embryonic dorso-ventral polarity in Drosophila. Homology of easter proteinase with Limulus proclotting enzyme and translational activation of Toll receptor synthesis.

The generation of dorso-ventral polarity during Drosophila embryogenesis is regulated by the action of 12 maternally expressed gene products, the dorsal group. These products act together to form a dorso-ventral nuclear gradient of the transcription factor dorsal. At least three of the dorsal group genes (snake, easter and gastrulation defective) encode secreted serine proteinases which probably function during early development in the perivitelline compartment of the embryo. Here, we report that the easter proteinase is homologous in its light chain sequence to the haemocyte proclotting enzyme (PCE) of the Japanese horseshoe crab Tachypleus tridentatus. PCE is the terminal member of a proteolytic cascade activated in response to microbial polysaccharides and acts to cleave coagulogen, an invertebrate equivalent of fibrinogen. On the basis of this homology we are able to predict with confidence the overall primary structure of the easter proteinase, its mode of activation and its substrate specificity. The result also suggests that easter functions zygotically in haemocytes in a Drosophila defence response analogous to that found in Tachypleus. We also show here that the Toll receptor protein is absent in early cleavage embryos but accumulates rapidly at the syncitial blastoderm stage, the developmental stage at which its function is required. This finding suggests that translation of Toll mRNA is regulated in response to fertilisation and egg deposition. These two observations are consistent with a model of dorso-ventral pattern formation in which a proteolytic cascade is activated uniformly in the perivitelline compartment of the embryo and causes the release of ventrally localised ligands of the Toll receptor. A possible alternative model in which a proteolytic cascade is activated in response to a ventrally restricted signal is also discussed.

Sequence and expression of LRR47, a novel embryonic leucine rich repeat protein of Drosophila.

Leucine-rich repeats (LRRs) are 22-28 amino acid long sequence motifs found in a variety of extracellular, membrane and cytoplasmic proteins. They are believed to mediate specific protein-protein interactions and to function in cellular adhesion. In Drosophila, four LRR proteins are known and each plays an important role in embryogenesis. In this paper we report the cloning of a cDNA that encodes a fifth Drosophila embryonic LRR protein, LRR47. The sequence includes a hydrophobic N-terminal which may constitute an ER signal sequence, eight LRR copies and a unique C-terminal. The transcript of the LRR47 gene is detected in adult females and in early embryogenesis. It is not found in adult males and is only present at low levels in embryos after 6 h of development. In Western blot experiments, a protein of approx. 47 kDa, which is expressed in a similar developmental profile and purifies in peripheral membrane protein extracts, is detected by an antibody specific for LRR47. The LRR47 gene maps to position 32A on the left arm of chromosome 2, an interval in which three genes with semi-lethal maternal effects (dal, hup and wdl) are located.

Drosophila ribosomal protein L18a: cDNA sequence, expression and chromosomal localization of the gene.

We describe the cDNA sequence of the Drosophila homologue of the rat ribosomal protein L18a. The protein sequence predicted has identical or conservatively substituted amino acids in 80% of positions. It is distinctly basic in character with an overall net positive charge of + 20. Analysis of L18a RNA with the Northern blot technique shows it to be expressed both during embryonic development and in the adult fly. In situ hybridisation to polytene chromosomes reveals that the L18a gene(s) is located at 54B on the second chromosome.

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Promoter sequence and expression of the leucine-rich repeat gene LRR47: evidence for cytoplasmic and nuclear localization in Drosophila embryos and cells.

In Drosophila, proteins containing leucine-rich repeats (LRR) play diverse roles during embryonic development. In particular, they function in cell adhesion and cellular signalling and have in common the ability to mediate reversible protein-protein interactions. The sequence and chromosomal location of Drosophila LRR47, which encodes a protein with eight LRR repeats, were reported previously. In this paper the 5' flanking region of the LRR47 gene is described and the initiation point for the maternal transcription unit is defined. LRR47 belongs to a subfamily of LRR proteins that have in common the ability to interact with ras GTPase. Whole-mount in situ hybridization studies show that the LRR47 transcript is uniformly distributed in early cleavage embryos but becomes depleted at the termini by the blastoderm stage. There is a specific requirement for ras function in the embryonic termini at this developmental stage. The distribution of LRR47 protein in embryos and tissue culture cells was also studied. The protein is present in both the cytoplasm and nuclei of embryos until gastrulation and is seen to persist in the nuclei of the amnioserosa until later stages of development. The protein is also constitutively present in growing SL2 culture cells and again is present in both cytoplasm and nuclei. These results suggest that LRR47 function may be modulated in the cell or nuclear division cycle.

Characterisation of the gene for Drosophila amphiphysin.

A sequence similarity search of the Drosophila nucleotide database using vertebrate amphiphysin as a query identified a cDNA that encodes a Drosophila amphiphysin. The predicted protein has conserved sequence domains that should enable it to dimerise and bind to dynamin. Structural modelling suggests that the Src-homology-3 (SH3) domains of vertebrate and Drosophila amphiphysins are highly similar, supporting the putative ability of the latter to bind dynamin. However, the fly amphiphysin shows less conservation to sequences in the vertebrate amphiphysins that bind other endocytic components such as clathrin, AP-2 and endophilin. Amphiphysin is a single-copy gene that maps to position 49B on polytene chromosomes. Messenger RNA of this amphiphysin is expressed widely during embryogenesis and has elevated expression in a number of sites including the foregut, hindgut and epidermis, but not in the central nervous system. Taken together, these data are consistent with a role for Drosophila amphiphysin in endocytosis, but the details of this role may differ from that of vertebrate amphiphysins.

The Drosophila ankyrin repeat protein cactus has a predominantly alpha-helical secondary structure.

The cactus protein is the Drosophila homologue of the mammalian I kappa B family of cytoplasmic anchor proteins. We have expressed in E. coli and purified a cactus fusion protein, CACT-Bgl. CACT-Bgl protein contains the six ankyrin repeat sequences which are necessary for specific binding to the Drosophila rel family transcription factor dorsal. We show that the purified CACT-Bgl protein can bind specifically to dorsal and, using circular dichroism spectroscopy, that the protein adopts a largely alpha-helical secondary structure. A further analysis of the ankyrin repeat domains of cactus, using an improved secondary structure prediction program indicates that the N-terminal of the repeat will form into a loop structure and the C-terminal section into an interrupted, amphipathic alpha-helix. On the basis of these findings we propose that the ankyrin repeats of cactus fold together into helical bundles interconnected by diverged loops.

Wild type and constitutively activated forms of the Drosophila Toll receptor have different patterns of N-linked glycosylation.

Toll is a Drosophila membrane protein related in sequence to the mammalian platelet glycoprotein 1B and to the interleukin-1 receptor. It mediates a signal transduction pathway leading to the development of dorsoventral polarity in the Drosophila embryo. In this paper we show that a constitutively activated mutant receptor, Toll10B, is processed into a distinct isoform of slower electrophoretic mobility when compared with the wild type molecule in both cell lines and the embryo. The wild type protein can also be processed into this form if over-expressed but in the embryo is present as the smaller species. We show that the decrease in the mobility of Toll10B and over-expressed wild type receptors is caused by altered patterns of N-linked glycosylation and that both forms are secreted to the cell surface. On the basis of these results, we propose that the Toll10B receptor is unable to associate with a limiting co-factor which when bound directly or indirectly masks supplementary N-linked glycosylation sites.

Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IkappaB homologue cactus.

Cactus protein is a Drosophila homologue of the mammalian IkappaB family of cytoplasmic anchor proteins. In unstimulated cells they function to retain rel/NFkappaB transcription factors in the cytoplasm but are rapidly degraded in response to signalling. The destruction of cactus or IkappaBalpha allows the rel/NFkappaB transcription factor to relocalise to the nucleus. Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain. In this paper we show that, like mammalian IkappaBalpha, the PEST domain of cactus is phosphorylated by casein kinase II. We have localised the site of modification to a single residue, Ser468, and find no evidence for additional phosphorylation sites. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis.

A leucine-rich repeat peptide derived from the Drosophila Toll receptor forms extended filaments with a beta-sheet structure.

Leucine-rich repeats (LRRs) are 22-28 amino acid-long sequence motifs found in a family of cytoplasmic, membrane and extracellular proteins. There is evidence that LRRs function in signal transduction, cellular adhesion and protein-protein interactions. Here we report unusual properties of a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide form an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed.

X-ray diffraction and far-UV CD studies of filaments formed by a leucine-rich repeat peptide: structural similarity to the amyloid fibrils of prions and Alzheimer's disease beta-protein.

The development of neuro-degenerative diseases often involves amyloidosis, that is the formation of polymeric fibrillar structures from normal cellular proteins or peptides. For example, in Alzheimer's disease, a 42 amino acid peptide processed from the amyloid precursor protein forms filaments with a beta-sheet structure. Because of this, the structure and dynamics of polymeric peptide filaments is of considerable interest. We showed previously that a 23 amino acid peptide constituting a single leucine-rich repeat (LRRN) polymerises spontaneously in solution to form long filaments of a beta-sheet structure, a property similar to that of Alzheimer's beta-amyloid and prion peptides. Here we report that a variant of LRRN in which a highly conserved asparagine residue is replaced by aspartic acid does not form either filaments or beta structure. By contrast, a variant which replaces this asparagine residue with glutamine forms filaments ultrastructurally indistinguishable from those of LRRN. Electron micrographs of LRRN filaments show that many consist of two interleaved strands which appear to have a ribbon-like morphology. X-ray diffraction patterns from oriented LRRN fibres reveal that they are composed of long beta-sheet arrays, with the interstrand hydrogen bonding parallel to the filament axis. This 'cross-beta' structure is similar to that adopted by beta-amyloid and prion derived fibres. Taken together, these results indicate that the LRR filaments are stabilised by inter- or intra-strand hydrogen bonded interactions comparable to the asparagine ladders of beta-helix proteins or the 'glutamine zippers' of poly-glutamine peptides. We propose that similar stabilising interactions may underlie a number of characterised predispositions to neuro-degenerative diseases that are caused by mutations to amide residues. Our finding that amyloid-like filaments can form from a peptide motif not at present correlated with degenerative disease suggests that a propensity for beta-filament formation is a common feature of protein sub-domains.

A model of long-term decline in the transmissibility of an infectious disease: implications for the incidence of hepatitis A.

BACKGROUND: The epidemiology of hepatitis A in countries across the world is changing due to improvements in hygiene and living conditions which reduce the transmissibility of the infection. METHODS: A mathematical model is formulated to describe the changes in incidence of a directly transmitted infection produced by a long term decline in its transmissibility. The basic reproduction number, the parameter describing transmissibility, is considered as a function of time. The relationship between the basic reproduction number and the force of infection is derived. RESULTS: Theoretical examples demonstrate that a decline in transmissibility results in an initial decline in the force of infection, but that this may be followed by a substantial resurgence. Resurgences may be possible after several decades of declining incidence, and are most marked following a rapid decline. CONCLUSIONS: Countries which have experienced a rapid decline in the incidence of hepatitis A may be at risk of a resurgence. More detailed mathematical models, informed with data from regular age-stratified serological surveys, should provide the basis for decisions on vaccination policy.

Modeling measles, mumps, and rubella: implications for the design of vaccination programs.

Mathematical models of disease transmission are being used increasingly in the design of population-based vaccination programs. Their use is illustrated in a review of some modeling studies that have implications for the use of measles, mumps, and rubella vaccine. Investigations of vaccination strategy options yield predictions for effectiveness and cost-effectiveness analyses. A quantitative understanding of the factors affecting disease transmission enables the setting of targets for vaccination programs and underpins disease elimination initiatives.

The prevalence of hepatitis C in England and Wales.

OBJECTIVES: To estimate the background population prevalence of hepatitis C in England and Wales, observe the prevalence over time and assess the extent of infection outside of known risk groups. METHODS: Sera from residual specimens from adult patients submitted to laboratories in England and Wales were tested for anti-HCV. Testing was carried out using a cost-effective pooling strategy. RESULTS: Although the prevalence of anti-HCV was highest in 1986 (1.07%), in the multivariable analysis, prevalence did not vary significantly between the 3 periods 1986, 1991 and 1996 (P=0.14). The prevalence of infection was higher in males than in females (P=0.0013). An age-period-cohort analysis revealed a cohort effect due to a lower HCV prevalence in the most recent birth cohorts, that is, those born between the calendar years 1971-1975 and 1976-1980. CONCLUSIONS: The majority of HCV infections in England and Wales were probably acquired before 1986. Infections in younger males identified in 1996 may signify more recent acquisition by injecting drug use.

A mathematical model to measure the impact of the Measles Control Campaign on the potential for measles transmission in Australia.

BACKGROUND: The aims of this study were to determine the impact of the Australian Measles Control Campaign (MCC) on the transmission dynamics of measles by calculating the reproductive number (R) before and after the MCC, and to predict measles control in Australia in the future. METHODS: A national serosurvey was conducted before and after the MCC. Sera were tested for anti-measles IgG using enzyme immunoassay (EIA). A mathematical model, using serosurvey results and vaccine coverage estimates, was used to calculate the change in R after the MCC. RESULTS: The values of R calculated before and after the MCC were 0.90 and 0.57. At vaccine coverage levels indicated by the Australian Childhood Immunisation Register (ACIR), the value of R will exceed 1 (the epidemic threshold) in 2007-2008 nationally, and sooner in some regions of Australia. Coverage of at least 84% with two doses of MMR is required to sustain measles control. CONCLUSIONS: The Australian MCC had a significant impact on the transmission dynamics of measles. However, current vaccine coverage levels may result in indigenous measles transmission by 2007. Sustained efforts are required to improve coverage with two doses of MMR and to ensure elimination of indigenous measles transmission.