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Lamotrigine (LTG) is an important antiepileptic drug for the treatment of seizures in pregnant women with epilepsy. However, it is not known if the transport of LTG into placental cells occurs via a carrier-mediated pathway. The aim of this study was to investigate the uptake properties of LTG into placental cell lines (BeWo and JEG-3), and to determine the involvement of organic cation transporters (OCTs, SLC22A1–3) and organic cation/carnitine transporter (OCTNs, SLC22A4–5) in the uptake process. The uptake of LTG at 37 °C was higher than that at 4 °C. OCT1 and OCTNs were detected in both cell lines. The uptake of LTG was not greatly affected by the extracellular pH, Na+-free conditions, or the presence of l-carnitine, suggesting that OCTNs were not involved. Although several potent inhibitors of OCTs (chloroquine, imipramine, quinidine, and verapamil) inhibited LTG uptake, other typical inhibitors had no effect. In addition, siRNA targeted to OCT1 had no significant effect on LTG uptake. The mRNA expression in human term placenta followed the order OCTN2 > OCT3 > OCTN1 > OCT1 ≈ OCT2. These observations suggested that LTG uptake into placental cells was carrier-mediated, but that OCTs and OCTNs were not responsible for the placental transport process.

The nematode Caenorhabditis eleganshas a single gene, lev-11, encoding multiple tropomyosin isoforms. Two seventh exons are alternatively selected in the head and body regions of muscle cells and differentially affect muscle contractility in the respective regions.

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In the nematode somatic gonad, nonmuscle myosin and muscle myosin form distinct filaments and coordinate ovulatory contraction of the myoepithelial sheath. Nonmuscle myosin regulatory light chain is phosphoregulated, and its phosphorylation and dephosphorylation are critical for successful ovulation.

Two UNC-87 isoforms with seven calponin-like repeats are expressed widely in muscle and nonmuscle cells in Caenorhabditis elegans. They bind to actin and myosin and inhibit actomyosin motility in vitro. unc-87 mutation enhances contraction of nonstriated muscle in vivo, suggesting that UNC-87 isoforms are negative regulators of actomyosin contractility.

Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78–null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1–null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.

AIP1 is a conserved enhancer of ADF/cofilin-dependent actin dynamics. Caenorhabditis eleganshas two AIP1 genes. They have overlapping functions, and ablation of both AIP1 isoforms causes embryonic lethality with strong defects in the assembly of muscle sarcomeres.

Tardigrades, also known as water bears, have somatic muscle fibers that are responsible for movement of their body and legs. These muscle fibers contain thin and thick filaments in a non-striated pattern. However, the regulatory mechanism of muscle contraction in tardigrades is unknown. In the absence of extensive molecular and genomic information, we detected a protein of 31 kDa in whole lysates of tardigrades that cross-reacted with the antibody raised against nematode troponin I (TnI). TnI is a component of the troponin complex that regulates actin-myosin interaction in a Ca2+-dependent and actin-linked manner. This TnI-like protein was co-extracted with actin in a buffer containing ATP and EGTA, which is known to induce relaxation of a troponin-regulated contractile system. The TnI-like protein was specifically expressed in the somatic muscle fibers in adult animals and partially co-localized with actin filaments in a non-striated manner. Interestingly, the pharyngeal muscle did not express this protein. These observations suggest that the non-striated somatic muscle of tardigrades has an actin-linked and troponin-regulated system for muscle contraction.

Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegansmuscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction.

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal β-propeller, whereas one residue is located in the C-terminal β-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

Abstract: Kettin is a unique member of the connectin/titin family of muscle elastic proteins, which has repetitive immunoglobulin-like domains that are separated by weakly conserved linker sequences. In striated muscles of insects and crayfish, kettin binds to actin filaments and localizes to the Z-disc and its adjacent region in the I-band. Recent sequence analysis of invertebrate connectin/titin (also known as SLS proteins) has revealed that kettin is a splice variant of connectin/titin. In contrast, in the nematode Caenorhabditis elegans, the kettin gene is independent of the genes for other connectin/titin-related proteins. Immunofluorescent localization of kettin shows that it localizes to the I-bands in the obliquely striated body wall muscle. Therefore, C. elegans is an attractive model system to study specific functions of kettin in muscle cells.

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegansprotein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.

We studied the potential role of the Duffy antigen and glycophorin A as receptors for rodent malaria parasite invasion of erythrocytes. Parasitemia increased exponentially after infection with Plasmodium berghei NK65, P. chabaudi, and P. vinckei in Duffy antigen knockout, glycophorin A knockout, and wild-type mice, indicating that the Duffy antigen and glycophorin A are not essential for these malaria parasites. However, parasitemia of the Duffy antigen knockout mice infected with P. yoelii 17XL remained constant from day 5 to 14 after infection, and then decreased, resulting in autotherapy. The treatment of P. yoelii 17XL-infected Duffy antigen knockout mice with anti-CD4 antibody increased the parasitemia 15 days after infection and the mice eventually died, indicating that CD-4-positive cells play an important role in the clearance of P. yoelii 17XL at the late stage of the infection.

Ovulation in the nematode Caenorhabditis elegansis coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegansreproductive system.

The effect of the poly(oxyethylene) (POE) chain length of ionic surfactants, expressed as C12H25-(OCH2CH2)-nOCH2C6H3(OH)(NO2) (n= 2—9), on the extraction of monovalent cations, including alkali metal and tetraalkylammonium (TAA) ions, was investigated. The extraction constants (Kex) with surfactants having a POE chain of n= 6 were almost constant for each alkali metal ion. The Kexsequence was Li+< Na+< Cs+= K+= Rb+. The extractability and selectivity largely decreased as nin the surfactant decreased (n< 6). The effect of the POE chain length was related to the stability of the alkali metal-POE complex. Unlike an alkali metal ion, the Kexvalues for all TAA ions were almost constant with surfactants of n= 2—8. These facts indicated that the surfactant extracts alkali metal ions by both complex formation with its POE chain and ion-pair formation with a terminal p-nitrophenolate anion. On the other hand, TAA ions were extracted simply by ion-pair formation with a p-nitrophenolate anion of the surfactant. The proposed structures are discussed from the viewpoint of the spectrum shift of the extracted species.

Members of the transforming growth factor β (TGF‐β) family mediate key events in cell growth and development. Various receptors for diverse members of the TGF‐β family have recently been isolated and sequenced. These receptors form a family (TβR family) with a Ser/Thr kinase domain in common. To understand the divergence pattern of the TβR family during animal evolution, we have conducted cloning of cDNAs encoding the TβR family members from Ephydatia fluviatilis, a freshwater sponge. We obtained seven cDNAs (sALK‐1–sALK‐7) which are closely related in structure to known family members. Including these sponge sequences, a phylogenetic tree of the family members was inferred by a maximum likelihood method. The phylogenetic tree suggests that the sponge receptors sALK‐1–sALK‐3, which are closely related to each other, are sponge homologs of vertebrate activin type I receptor (ActR‐I). sALK‐5 is likely to be a homolog of TGF‐β type II receptor. sALK‐4 and sALK‐6 might be ancestral precursors of type I and type II receptors, respectively, and sALK‐7 is possibly an ancestral precursor of both types. The tree revealed that most, if not all, of the gene duplications that gave rise to known subtypes with distinct ligand specificities antedate the divergence of parazoans and eumetazoans, the earliest divergence of extant animal phyla.

Abstract.: To know whether genes involved in cell–cell communication typical of multicellular animals dramatically increased in concert with the Cambrian explosion, the rapid evolutionary burst in the major groups of animals, and whether these genes exist in the sponge lacking cell cohesiveness and coordination typical of eumetazoans, we have carried out cloning of the G-protein α subunit (Gα) and the protein tyrosine kinase (PTK) cDNAs from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We obtained 13 Gα and 20 PTK cDNAs. Generally animal gene families diverged first by gene duplication (subtype duplication) that gave rise to diverse subtypes with different primary functions, followed by further gene duplication in the same subtype (isoform duplication) that gave rise to isoform genes with virtually identical function. Phylogenetic trees of Gα and PTK families including cDNAs from sponge and hydra revealed that most of the present-day subtypes had been established in the very early evolution of animals before the parazoan–eumetazoan split, the earliest branching among the extant animal phyla, by extensive subtype duplication: for PTK and Gα families, 23 and 9 subtype duplications were observed in the early stage before the parazoan–eumetazoan split, respectively, and after that split, only 2 and 1 subtype duplications were found, respectively. After the separation from arthropods, vertebrates underwent frequent isoform duplications before the fish–tetrapod split. Furthermore, rapid amino acid changes appear to have occurred in concert with the extensive subtype duplication and isoform duplication. Thus the pattern of gene diversification during animal evolution might be characterized by bursts of gene duplication interrupted by considerably long periods of silence, instead of proceeding gradually, and there might be no direct link between the Cambrian explosion and the extensive gene duplication that generated diverse functions (subtypes) of these families.

Abstract.: Protein tyrosine phosphatases (PTPs) regulate various physiological events in animal cells. They comprise a diverse family which are classified into two categories, receptor type and nonreceptor type. From the domain organization and phylogenetic tree, we have classified known PTPs into 17 subtypes (9 receptor-type and 8 nonreceptor-type PTPs) which are characterized by different organization of functional domain and independent cluster in tree. The receptor type PTPs are thought to be implicated in cell–cell adhesion by association of cell adhesion molecules. Since sponges are the most primitive multicellular animals and are thought to be lacking cell cohesiveness and coordination typical of eumetazoans, cloning and sequencing of PTP cDNAs of Ephydatia fluviatilis (freshwater sponge) have been conducted by RT-PCR to determine whether or not sponges have PTP genes in their genomes. We have isolated nine PTPs, of which five are possibly receptor type. A phylogenetic tree including the sponge PTPs revealed that most of the gene duplications that gave rise to the 17 subtypes had been completed in the very early evolution of animals before the parazoan–eumetazoan split, the earliest branching among extant animal phyla. The family tree also revealed the rapid evolutionary rate of PTP subtypes in the early stage of animal evolution.

The animal cyclic nucleotide phosphodiesterases (PDEs) comprise at least seven subtypes, PDE1–7, which differ from each other in domain organization and primary function, and they diverged from an ancestral gene by gene duplication and domain shuffling during animal evolution. To obtain rough estimates for the divergence times of these subtypes, cloning of PDE cDNAs from Ephydatia fluviatilis(freshwater sponge) by RT‐PCR was carried out. We obtained four cDNAs, EFPDE1, EFPDE2, EFPDE3, and EFPDE4, which are possibly homologs of the vertebrate PDE1, PDE2, PDE3, and PDE4, respectively, judging from the sequence similarity, domain organization, and branching pattern in the phylogenetic tree. The phylogenetic tree of the PDE family revealed that most gene duplications and domain shufflings that gave rise to different subtypes had been completed in the early evolution of animals before the separation of sponges and eumetazoans.

To know whether or not the set of genes involved in the inositol phospholipid signaling pathway already existed in the early evolution of animals, we carried out cloning of cDNAs encoding phospholipase Cs (PLCs) from Ephydatia fluviatilis(freshwater sponge) and Hydra magnipapillatastrain 105 (hydra). We isolated two PLC cDNAs, PLC‐βS and PLC‐γS, from sponge and three cDNAs, PLC‐βH1, PLC‐βH2, and PLC‐δH, from hydra. From the domain organization and the divergence pattern in the PLC family tree, the sponge PLC‐βS and PLC‐γS and the hydra PLC‐δH are possibly homologous to the vertebrate PLC‐β, PLC‐γ and PLC‐δ subtypes, respectively. A detailed phylogenetic analysis suggests that the hydra PLC‐βH1 and PLC‐βH2 are homologs of the vertebrate PLC‐β1/2/3/DrosophilaPLC21 and the vertebrate PLC‐β4/Drosophila norpA, respectively. A phylogenetic analysis of the PLC family and the protein kinase C (PKC) family, together with that of the G protein α subunit (Gα) family, revealed that the origin of the set of genes Gαq, PLC, PKC involved in the inositol phospholipid signaling pathway is very old, going back to dates before the parazoan‐eumetazoan split, the earliest branching among extant animal phyla.

The protein tyrosine kinases (PTKs) are a large protein family consisting of many subfamilies with a variety of domain structures. The basic functions are thought to differ for different subfamilies. To know the dates at which the subfamilies diverged by gene duplications, a phylogenetic tree of the PTKs was inferred by comparing sequences from a wide range of species covering diploblasts and triploblasts. The PTK tree revealed that almost all of the gene duplications that gave rise to different subfamilies occurred rapidly before the diploblast–triploblast split, accompanying with rapid amino acid substitutions. This type of gene duplication was, however, rarely observed after that split. Long after the subfamily divergence, another type of gene duplication that gave rise to diverse tissue‐specific genes occurred in each subfamily on the chordate lineage since the separation from arthropods. This type of gene duplication occurred frequently before the fish–tetrapod split, accompanying with rapid amino acid substitutions. In contrast, both the frequency of gene duplications and the rate of the amino acid substitutions were considerably reduced after that split. These results strongly suggest that the PTKs diverged intermittently, but not gradually, during animal evolution.

Eight compounds of type (II) are prepared as precursors of the alkaloids from benzaldoxime methyl ethers of type (I) by the title reaction as key step.

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