Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.     

Fascins, and their roles in cell structure and function

The fascins are a structurally unique and evolutionarily conserved group of actin cross-linking proteins. Fascins function in the organisation of two major forms of actin-based structures: dynamic, cortical cell protrusions and cytoplasmic microfilament bundles. The cortical structures, which include filopodia, spikes, lamellipodial ribs, oocyte microvilli and the dendrites of dendritic cells, have roles in cell-matrix adhesion, cell interactions and cell migration, whereas the cytoplasmic actin bundles appear to participate in cell architecture. We discuss the current understanding of the cellular mechanisms that regulate the binding of fascin to actin and how these processes contribute to the organisation or disassembly of cell protrusions. Although the in vivo roles of fascin have been studied principally in Drosophila, several human diseases are associated with inherited or acquired alterations in the expression of fascins. Strategies to modulate fascin-containing protrusions and thereby cell adhesive and migratory behaviour could have potential for therapeutic intervention in these conditions. The supplementary material referred to in this section can be found at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2002/v24.350.html     

Automated detection of remote homology

The classification of a newly identified protein as a member of a superfamily is important for focusing experiments on its most likely functions. Such classification, often performed by hand, has now been fully automated. This sophisticated new approach takes into account not only alignment scores but also a number of other computable attributes, such as functional sites deduced from sequence conservation patterns.     

Going to the roots of the stem cell controversy

The purpose of this paper is to describe the scientific background to the current ethical and legislative debates about the generation and use of human stem cells, and to give an overview of the ethical issues underlying these debates.
The ethical issues discussed are 1) stem cells and the status of the embryo, 2) women as the sources of ova for stem cell production, 3) the use of ova from other species, 4) slippery slopes towards reproductive cloning, 5) the public presentation of stem cell research and 6) the evaluation of scientific uncertainty and its implications for public policy.     

Characterization of a novel testicular form of human hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.     

Ligand Binding and Polymerization Characteristics of Human Milk Folate Binding Protein Depend on Concentration of Purified Protein and Presence of Amphiphatic Substances

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.     

Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack.     

Molecular phylogeny of the kelch-repeat superfamily reveals an expansion of BTB/kelch proteins in animals

Background  

The kelch motif is an ancient and evolutionarily-widespread sequence motif of 44–56 amino acids in length. It occurs as five to seven repeats that form a β-propeller tertiary structure. Over 28 kelch-repeat proteins have been sequenced and functionally characterised from diverse organisms spanning from viruses, plants and fungi to mammals and it is evident from expressed sequence tag, domain and genome databases that many additional hypothetical proteins contain kelch-repeats. In general, kelch-repeat β-propellers are involved in protein-protein interactions, however the modest sequence identity between kelch motifs, the diversity of domain architectures, and the partial information on this protein family in any single species, all present difficulties to developing a coherent view of the kelch-repeat domain and the kelch-repeat protein superfamily. To understand the complexity of this superfamily of proteins, we have analysed by bioinformatics the complement of kelch-repeat proteins encoded in the human genome and have made comparisons to the kelch-repeat proteins encoded in other sequenced genomes.     

Rapid automatic detection and alignment of repeats in protein sequences

Many large proteins have evolved by internal duplication and many internal sequence repeats correspond to functional and structural units. We have developed an automatic algorithm, RADAR, for segmenting a query sequence into repeats. The segmentation procedure has three steps: (i) repeat length is determined by the spacing between suboptimal self-alignment traces; (ii) repeat borders are optimized to yield a maximal integer number of repeats, and (iii) distant repeats are validated by iterative profile alignment. The method identifies short composition biased as well as gapped approximate repeats and complex repeat architectures involving many different types of repeats in the query sequence. No manual intervention and no prior assumptions on the number and length of repeats are required. Comparison to the Pfam-A database indicates good coverage, accurate alignments, and reasonable repeat borders. Screening the Swissprot database revealed 3,000 repeats not annotated in existing domain databases. A number of these repeats had been described in the literature but most were novel. This illustrates how in times when curated databases grapple with ever increasing backlogs, automatic (re)analysis of sequences provides an efficient way to capture this important information.     

Evaluation of the performance enhancement of silicone biofouling-release coatings by oil incorporation

In response to increased evidence of ecosystem damage by toxic antifouling paints, many researchers have developed nontoxic silicone fouling release coatings. The fouling release capability of these Systems may be improved by adding nonbonding silicone oils to the coating matrix. This idea has been tested by comparing the adhesion strength of hard- and soft-fouling organisms on a cured polydimethylsilicone (PDMS) network to that of the same network containing free polydi-methyldiphenylsilicone (PDMDPS) oil at five exposure sites in North America and Hawaii. Fouling coverage is discussed, together with the bioadhesion data, to emphasize that although these coatings foul the fouling is easily removed. The partitioning of the incorporated oil upon exposure of the coatings to a simulated marine environment containing sediment was determined. Less than 1.1 wt% of the incorporated oil was lost from the coating over one year, and the toxicity of these coatings was shown to be minimal to shrimp and fish. Brush abrasion wear was greater for coatings containing free oil, but the modulus of elasticity was not appreciably decreased by the addition of 10wt% free oil.