Fe(CO)4 and related compounds as isolobal fragments.

The M(CO)(4) fragment can be assigned to be isolobal with both CH(3)(+) and CH(2). In order to investigate this ambiguous isolobal assignment, we report calculations on compounds of the type M(CO)(4)L(n), where M is Fe (n = 0), Mn (n = -1), and Co (n = +1) and L is an η(2) ligand with a π bond, generally an alkene. The L's are varied in electron-withdrawing ability, and patterns in computed structural parameters are investigated. We report that the equatorial OC-M-CO angle is sensitive to the electron-withdrawing ability of the alkene just as the isolobal prediction suggests. Other structural parameters that vary monotonically with electron-withdrawing ability of the alkene are the "bending back" of the alkene, the metal-ligand bond distances, and carbon-oxygen bond distances. Changing the metal from neutral Fe to a negatively charged Mn or positively charged Co has the result of increasing and decreasing, respectively, the OC-M-CO angle. Several compounds of Ni(CO)(3)L are also investigated as a further example of the ability of the isolobal concept to yield chemically useful information.

Heme Aggregation inhibitors: antimalarial drugs targeting an essential biomineralization process.

Malaria, resulting from the parasites of the genus Plasmodium, places an untold burden on the global population. As recently as 40 years ago, only 10% of the world's population was at risk from malaria. Today, over 40% of the world's population is at risk. Due to increased parasite resistance to traditional drugs and vector resistance to insecticides, malaria is once again resurgent. An emergent theme from current strategies for the development of new antimalarials is that metal homeostasis within the parasite represents an important drug target. During the intra-erythrocytic phase of its life cycle, the malaria parasite can degrade up to 75% of an infected cell's hemoglobin. While hemoglobin proteolysis yields requisite amino acids, it also releases toxic free heme (Fe(III)PPIX). To balance the metabolic requirements for amino acids against the toxic effects of heme, malaria parasites have evolved a detoxification mechanism which involves the formation of a crystalline heme aggregate known as hemozoin. An overview of the biochemistry of the critical detoxification process will place it in the appropriate context with regards to drug targeting and design. Quinoline-ring antimalarial drugs are effective against the intraerythrocytic stages of pigment-producing parasites. Recent work on the mechanism of these compounds suggests that they prevent the formation of hemozoin. Evidence for such a mechanism is reviewed, especially in the context of the newly reported crystal structure of hemozoin. Additionally, novel drugs, such as the hydroxyxanthones, which have many of the characteristics of the quinolines are currently being investigated. Recent work has also highlighted two classes of inorganic complexes that have interesting antimalarial activity: (1) metal-N(4)O(2) Schiff base complexes and (2) porphyrins. The mechanism of action for these complexes is discussed. The use of these complexes as probes for the elucidation of structure-activity relationships in heme polymerization inhibitor design and the loci of drug resistance is also detailed. As the biochemistry of the complicated interactions between host, parasite, and vector become better understood, the rationale for new antimalarial drug treatments will continue to improve. Clearly, the homeostasis of metal ions is a complicated biochemical process and is not completely understood. For the immediate future, it does, however, provide a clear target for the development of new and improved treatments for malaria.

The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme.

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis. Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tektl staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.

The Rib43a protein is associated with forming the specialized protofilament ribbons of flagellar microtubules in Chlamydomonas.

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins as tektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1. 7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an approximately 4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43a gene on linkage group III. Sequence analysis of the RIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens (genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.

Expression of ciliary tektins in brain and sensory development.

Many types of neural tissues and sensory cells possess either motile or primary cilia. We report the first mammalian (murine testis) cDNA for tektin, a protein unique to cilia, flagella, and centrioles, which we have used to identify related proteins and genes in sensory tissues. Comparison with the sequence database reveals that tektins are a gene family, spanning evolution from Caenorhabditis elegans (in which they correlate with touch receptor cilia) and Drosophila melanogaster, to Mus musculus and Homo sapiens (in which they are found in brain, retina, melanocytes, and at least 13 other tissues). The peptide sequence RPNVELCRD, or a variant of it, is a prominent feature of tektins and is likely to form a functionally important protein domain. Using the cDNA as a probe, we determined the onset, relative levels, and locations of tektin expression in mouse for several adult tissues and embryonic stages by Northern blot analysis and in situ hybridization. Tektin expression is significant in adult brain and in the choroid plexus, the forming retina (primitive ependymal zone corresponding to early differentiating photoreceptor cells), and olfactory receptor neurons of stage embryonic day 14 embryos. There is a striking correlation of tektin expression with the known presence of either motile or primary cilia. The evolutionary conservation of tektins and their association with tubulin in cilia and centriole formation make them important and useful molecular targets for the study of neural development.

Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles.

Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3-4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.

Structural comparison of tektins and evidence for their determination of complex spacings in flagellar microtubules.

Recent structural studies indicate that a tektin heteropolymer forms a unique protofilament of flagellar microtubules. We report here the sequence of tektin C (approximately 47 kDa), predicted from its cDNA (GenBank U38523), compared to tektins A (approximately 53 kDa) and B (approximately 51 kDa) from sea urchin (Strongylocentrotus purpuratus) sperm flagellar microtubules, and compared to partial sequences reported from mouse and human. We are now able to make several observations concerning the tektin family: (1) their common structural features, (2) a comparison of their structure to intermediate filament proteins, and (3) their possible organization in the tektin filament polymer. The predicted amino acid sequence identities/similarities are: for tektins A and C, 42/54%, for tektins A and B, 34/51%; for tektins B and C, 29/42%; for tektin C and a partial cDNA clone from mouse testis, 55/65%; and for tektin B and a partial cDNA clone from the human brain, 45/47%. The three tektins (and the human clone) possess the exact sequence repeat RPNVELCRD. The structural pattern of all three tektin polypeptides is similar to intermediate filament proteins. Tektins are predicted to form extended rods composed of two alpha-helical segments (approximately 180 residues long) capable of forming coiled coils, which are interrupted by short non-helical linkers. The two segments are homologous in sequence and secondary structure, indicating a gene duplication event prior to the divergence of the three tektins. Along each tektin rod cysteine residues occur with a periodicity of approximately 8 nm, coincident with the axial repeat of tubulin dimers in microtubules. From EM data and calculations of secondary structure, the segment length of tektin AB heterodimers is likely to be 16 nm. Both segments of tektin C may be 24 nm long, but one may be 16 nm. On the basis of the available evidence, we propose that coassembly of tektin AB heterodimers with tektin C dimers produces filaments with overall repeats of 8, 16, 24, 32, 40, 48 and 96 nm, generating the basis for the complex spatial arrangements of axonemal components.

Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis.

Previous studies have shown that tektin A, one of three integral filamentous protein components of outer doublet microtubules, is synthesized in sea urchins in an amount correlating to the length of embryonic cilia initially assembled or experimentally regenerated. To investigate further the molecular mechanism for the regulation of tektin synthesis, tektin cDNA clones were used to assess mRNA levels during ciliogenesis, zinc-induced animalization, deciliation-induced regeneration and theophylline-induced elongation. Possibly involved in centriole replication, low, near-constant levels of mRNA for all three tektins are present in the unfertilized egg and during cleavage stages. Preceded by new synthesis of tektin B and C mRNAs, tektin A mRNA is up-regulated during ciliogenesis, but only tektin A mRNA levels correlate directly with ciliary length in animalized embryos; the others augment larger, non-limiting pools of tektins B and C. Tektin mRNAs decrease to steady-state levels after ciliogenesis, but are up-regulated again when the embryos are deciliated, correlating with the length of cilia to be deployed. In a species where a 3-fold ciliary length increase can be induced by theophylline treatment of zinc-arrested embryos, the mRNAs accumulate to proportionately higher levels during arrest but are not translated until induction, whereupon they decrease inversely with ciliary elongation. This suggests transcriptional control with respect to mRNA amounts but post-transcriptional control with respect to the expression of this phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

At least one of the protofilaments in flagellar microtubules is not composed of tubulin.

BACKGROUND: The core of the eukaryotic flagellum is the axoneme, a complex motile organelle composed of approximately 200 different polypeptides. The most prominent components of the axoneme are the central pair and nine outer doublet microtubules. Each doublet microtubule contains an A and a B tubule; these are composed, respectively, of 13 and 10-11 protofilaments, all of which are thought to be made of tubulin. The mechanisms that control the assembly of the doublet microtubules and establish the periodic spacings of associated proteins, such as dynein arms and radial spokes, are unknown. Tektins, a set of microtubule-associated proteins, are present in the axoneme as stable filaments that remain after the extraction of doublet microtubules; they are localized near to where the B tubule attaches to the A tubule and near to the binding sites for radial spokes, inner dynein arms and nexin links. Tektin filaments may contribute in an interesting way to the structural properties of axonemes. RESULTS: We have fractionated doublet microtubules from sea urchin sperm flagella into ribbons of stable protofilaments, which can be shown to originate from the A tubule. Using cryo-electron microscopy, conventional electron microscopy, scanning transmission electron microscopy, three-dimensional reconstruction and kinesin decoration, we have found that one protofilament in the ribbon is not composed of tubulin. This protofilament is an integral protofilament of the A tubule wall, has less mass per unit length than tubulin and does not bind kinesin. CONCLUSION: Contrary to what is generally assumed, at least one protofilament in the wall of the A tubule is not composed of tubulin. Our data suggest that this nontubulin protofilament is primarily composed of tektins, proteins that show some structural similarity to intermediate filament proteins. A 480 A axial periodicity within these ribbons, revealed by scanning transmission electron microscopy, can be related to the structure of tektin, and may determine the large-scale structure of the axoneme in terms of the binding of dynein, nexin and radial spokes to the doublet microtubule.

Tektins are heterodimeric polymers in flagellar microtubules with axial periodicities matching the tubulin lattice.

Tektins are proteins that copartition with tubulin in a stable ribbon of three protofilaments from ciliary and flagellar microtubules. After purification, tektins A, B, and C from sea urchin sperm flagellar microtubules appear as extended relatively insoluble filaments, < 5 nm in diameter. We used cross-linking reagents to investigate the associations and structural organization of subunits within tektin polymers isolated from stable protofilament ribbons of Strongylocentrotus purpuratus. We show by SDS-polyacrylamide gel electrophoresis, immunoblots, and transmission electron microscopy that tektins are continuous heteropolymers in the stable protofilament ribbons, and thus flagellar microtubules. Our results also provide evidence for the arrangement of different tektin polypeptides within "core" filaments containing equimolar tektins A and B. Treatment of these core filaments with bis(sulfosuccinimidyl)suberate and with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide yielded a predominant cross-linked approximately 106-kDa heterodimer of tektins A and B; similar results were obtained by glutaraldehyde cross-linking of tektins solubilized under mild conditions. Finally, cross-linking with 3,3'-dithiobis(sulfosuccinimidylpropionate) revealed a 16 nm periodicity in isolated tektin AB filaments that can be related to the 8 nm tubulin dimer lattice and to periodically associated microtubule components.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules.

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48-50 kDa in isolated spindles and centrosomes from CHO cells.

Tektin B1 from ciliary microtubules: primary structure as deduced from the cDNA sequence and comparison with tektin A1.

Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules, and they may also be present in centrioles, centrosomes and mitotic spindles. We report here the cloning and sequencing of a cDNA for ciliary tektin B1. Comparison of the predicted amino acid sequence of tektin B1 with the previously published sequence for tektin A1 reveals several features that better define this class of proteins. Like tektin A1, the central region of the tektin B1 polypeptide chain is predicted to form a coiled-coil rod, consisting of four major alpha-helical regions that are separated by non-helical linkers. Between the central rod domains of tektins A and B there is a 34%/20% amino acid sequence identity/similarity, including equivalent 50-residue segments containing 36 identities, and a high probability of long-range structural homology. The tektin polypeptide chains are divided into two major segments that have significant sequence homology to each other, both within a given tektin chain and between tektins A and B, indicative of gene duplication events. The tektins have a secondary structure and molecular design similar to, but a low primary sequence homology with, intermediate filament proteins. Unlike tektin A1, tektin B1 lacks any part of the C-terminal IFP consensus sequence.

Primary structure of tektin A1: comparison with intermediate-filament proteins and a model for its association with tubulin.

Tektins are proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules and that have biochemical and immunological properties similar to those of intermediate-filament proteins. We report here the sequence of a cDNA for tektin A1, one of the main tektins from Strongylocentrotus purpuratus sea urchin embryos. By hybridization analysis, tektin A mRNA appears maximally at ciliogenesis. The predicted structure of tektin A1 (M(r) 52,955) is a series of alpha-helical rod segments separated by nonhelical linkers. The two halves of the rod appear homologous and are probably related by gene duplication. Comparison of tektin A1 with intermediate-filament proteins, including nuclear lamins, reveals a low amino acid homology but similar molecular motif, i.e., pattern of helical and nonhelical domains. This study indicates that tektins are unique proteins but may be evolutionarily related to intermediate-filament proteins, and suggests a structural basis for the interaction of tektins and tubulin in microtubules.

Evidence for a non-tubulin spindle matrix and for spindle components immunologically related to tektin filaments.

Tektins were originally described as a set of three filamentous proteins (tektin A, B and C) associated with the walls of axonemal microtubules of sea urchin sperm. Using affinity-purified polyclonal antibodies raised against tektins of two sea urchin species, Lytechinus pictus and Strongylocentrotus purpuratus, we looked for tektin-like components in microtubule systems other than axonemes. By immunofluorescence microscopy we observed labeling of meiotic spindles in eggs of the surf clam Spisula solidissima and in several mammalian cell lines. In Spisula eggs the tektin-like antigens were still associated with the spindles after about 95% of the tubulin had been removed via a calcium/cold treatment. In pig kidney epithelial cells the tektin-like antigen appeared to be associated with bundles of calcium-stable spindle microtubules. By SDS-PAGE immunoblot the affinity-purified anti-tektins recognized several polypeptides in tubulin-depleted spindle remnants of Spisula eggs: A approximately 52 kDa, 1 M KCl-resistant component was identified by the antibody raised against tektin C from S. purpuratus, a approximately 48 kDa component was recognized by the antibody specific for tektin A from L. pictus, and three polypeptide bands (approximately 64 kDa, approximately 100 kDa and greater than 200 kDa) were detected by the antibody specific for tektin C from L. pictus. Only the latter antibody, however, stained Spisula spindles by immunofluorescence microscopy. We further report that the sensitivity of antibody recognition of proteins on immunoblots is dependent on the purity of sodium dodecyl sulfate.

Identification of a tektin-like protein associated with neurofilaments in the developing chick nervous system.

A 160-kD polypeptide, which is recognized by an affinity-purified polyclonal antibody to the 55-kD tektin-A polypeptide from sea urchin sperm flagellar microtubules, is associated with neurofilaments in embryonic chick nerve cells. Antibodies to tektin-A and monoclonal antibodies to the neurofilament triplet proteins colocalize to filaments in cultured nerve cells and to filaments in extracts of chick spinal cord, using indirect immunofluorescence microscopy and immunogold electron microscopy. The antigen reacting with anti-tektin-A in chick brain and spinal cord extracts has been identified as a 160-kD polypeptide by SDS-PAGE and has been shown to be distinct from the known neurofilament-triplet proteins by two-dimensional immunoblot analysis. These data suggest that a unique protein with limited sequence homology to tektin-A is a component of the neuronal cytoskeleton and is incorporated into or associated with neurofilaments.

Multiple immunoblot: a sensitive technique to stain proteins and detect multiple antigens on a single two-dimensional replica.

A multiple immunoblotting technique was developed to positively identify up to three different antigens on a single nitrocellulose replica of a two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel. Three highly sensitive immunoblot assays were selected, including: horseradish peroxidase/luminescence, alkaline phosphatase, and silver-enhanced immunogold. As a major advantage, the method permits a simultaneous detection of up to three different antigens without eluting the antibody-dye complex between staining of single polypeptides, thus providing a highly accurate identification of closely migrating components. The staining procedure is summarized in a flow chart. In addition to the multiple immunoblot staining, some suggestions are provided for a sensitive protein staining.

Retention of ciliary ninefold structure after removal of microtubules.

When axonemes of isolated gill cilia from the bay scallop Aequipecten irradians are heated at 45 degrees C for a minimum of 8 min in a 10 mM-Tris-HCl (pH 8), 1 mM-EDTA solution, nearly 80% of the tubulin is solubilized but most minor structural proteins are retained in a ninefold symmetrical configuration. This remnant consists of the junctional protofilaments, derived from outer doublet tubules, interconnected by nexin linkages, with radial spoke components still directed inwards. The remnant is of the same length as the original cilium, with the junctional protofilaments attached at the distal end to the ciliary tip and at the proximal end to the basal plate. Virtually identical fractionations can be achieved with blastula cilia isolated from both arctic and tropical sea-urchin embryos. The remnant is resistant to salt up to at least 1 M concentration, judged by the constancy of protein composition. Immunoblotting with antibodies against sea-urchin sperm flagellar tektins indicates that the tektins remain within the ciliary remnant, supporting their location within the junctional protofilament domain. The fractionation is inhibited by low pH, by magnesium or calcium ions in the millimolar range, and by monovalent ions at 10-fold higher concentrations. About a quarter of the total ciliary calmodulin is bound to the axoneme at micromolar calcium levels but most is released upon thermal fractionation. Polymerization of tubulin in the presence of the remnant results in singlet microtubules, separate from the remnant proper, suggesting that doublet formation may require coordinate co-assembly of tubulin with skeletal proteins. These observations demonstrate the existence of a fibrous skeleton in the axoneme, composed largely of ciliary tektins, nexin linkages, and other structural proteins.