Cloning and characterization of a novel radish protein kinase which is homologous to fungal cot-I like and animal Ndr protein kinases

According to the similarity of the amino acid sequences in their catalytic domains, eukaryotic protein kinases have been classified into the five main groups: 'AGC', 'CaMK', 'CMGC', 'PTK' and 'other'. The AGC group, represented by the cyclic nucleotide-dependent kinases (PKA and PKG), the calcium-phospholipid-dependent kinases (PKC) and the ribosomal S6 protein kinases, are poorly characterized in plants except for a few cases. In this study, in order to gain a better understanding of plant protein kinases in the AGC group, three cDNAs encoding novel protein kinases, RsNdr1 and RsNdr2a/b, were cloned from radish and characterized by molecular and biochemical methods. The deduced amino acid sequences of RsNdr1 and RsNdr2a/b contained all 12 conserved catalytic subdomains which are characteristic of the eukaryotic Ser/Thr protein kinases. A cell lysate from E. coli overexpressing RsNdr1 fusion protein had protein kinase activity toward a conventional protein substrate (myelin basic protein), whereas that from E. coli harboring a fusion plasmid encoding kinase-dead RsNdr1 or RsNdr2 did not show any protein kinase activity. A phylogenetic tree for 17 protein kinases from various organisms showed that the RsNdrs are more closely related to the protein kinases in a particular subgroup of the 'AGC' (fungal cot1-like and animal Ndr kinases) than to the authentic 'AGC' protein kinases, such as PKA, PKC or ribosomal S6 kinase.     

Molecular recognition in solid-state crystallization: colored chiral adduct formations of 1,1'-bi-2-naphthol derivatives and benzoquinone with a third component

Molecular recognition in solid-state crystallization involving derivatives of 1,1'-bi-2-naphthol and benzoquinone was studied. No adduct crystal was formed when crystals of biphenyl were further added as a third component to a grinding mixture of crystals of chiral 1,1'-bi-2-naphthol and benzoquinone, which by itself did not form an adduct. This contrasts with the case in which further addition of naphthalene crystals to the same mixture produced a new red crystal. Adduct formations using chiral 6,6'-dibromo-1,1'-bi-2-naphthol in place of 1,1'-bi-2-naphthol were also studied. In this case, adducts were produced either with or without biphenyl as a third compound, but the colors of the adducts differed significantly in the two cases: red and bluish-black. The same three-component adduct crystals were produced from solid-grinding and solution crystallization and the structure was determined by X-ray diffractometry. Based on the crystal structures, theoretical calculations were carried out to compare the mechanism of colorations in the binary and the ternary complexes.     

Estimation of the highest chromosome number of eukaryotes based on the minimum interaction theory

According to the minimum interaction theory, the chromosome evolution of eukaryotes proceeds as a whole toward increasing the chromosome number. This raises the following two questions: what was the starting chromosome number of eukaryotes and does the chromosome number increase infinitely? We attempted to provide a theoretical framework to resolve these questions. We propose that the species with n=2 observed in Protozoa, Platyhelminthes, Annelid, Algae, Fungi and higher plants would be chromosomal relicts conserving the karyotypes of ancestral eukaryotes. We also propose that the ideal highest number of eukaryotes (n(max)) can be given by an inverse of the minimum terminal interference distance (It(min)) in crossing-over (n(max)=100/It(min)). AsIt(min) =0.6 in mammals, n(max) approximately 166. On the other hand, the value estimated by computer simulations is somewhat lower with n(max)=133-138. Our arguments can be applied to other eukaryotes, if they have a localized centromere and the ratio of total synaptonemal complex/nuclear volume is comparable to that of mammals. We revealed that the index of gene shuffling per karyotypes (G) by means of the total number of gamete types with different gene combinations can be formulated asG =2(n+Fxi), where Fxi means interstitial chiasma frequency per cell corresponding to crossing-over mediated by the recombination nodule. The Fxi value increases in proportion to the n value in areas where n<40, but decreases gradually when n>40 and becomes zero when n>83. Therefore, in the ultimate karyotype with n(max)=166, FXi=0 andG =2(n)=2(166), where gene shuffling is guaranteed by the random orientation of chromosomes at the equatorial plate of meiotic metaphase I.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.     

Identification of a new multigene four-transmembrane family (MS4A) related to CD20, HTm4 and beta subunit of the high-affinity IgE receptor

We report here the cloning of eight new cDNAs that encode a family of proteins related to the B-cell-specific antigen CD20, a hematopoietic-cell-specific protein HTm4, and high affinity IgE receptor beta chain (FcvarepsilonRIbeta). They include four clones from human, and another four clones from mouse. They share similar structure (four transmembrane domains) with amino acid identities of 25-40%. Therefore, they represent distinct genes and comprise a gene superfamily. This superfamily is now named membrane-spanning four-domains, subfamily A (the approved symbol is MS4A) to distinguish them from tetraspanins with similar structure. The highest homologies among these proteins are found in the transmembrane domains, especially in the first and second transmembrane domains, and conserved residues are also recognized in the inter-transmembrane domains. In northern blot, they were mostly expressed in lymphoid tissues: thymus and spleen. However, some were expressed in nonlymphoid tissues including brain, heart, kidney, liver, testis, lung, GI tracts, and pancreas. They may represent proteins functioning either directly as ligand-gated ion channels or as essential components of such channels. The identification of this relatively large gene family in various tissues will allow the further elucidation of physiological significance of this gene family, that is currently unclear.     

Iba1 is an actin-cross-linking protein in macrophages/microglia

Iba1 is a 17-kDa EF hand protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. When exposed to macrophage colony-stimulating factor (M-CSF), microglia cell line MG5 immediately produces intense membrane ruffles in which Iba1 accumulates together with filamentous actin. In this study, we investigated the physical interaction between Iba1 and actin by centrifugation assay and electron microscopic examination and showed that Iba1 possesses actin-binding and -cross-linking activities. Inhibitory mutant Iba1 that suppresses M-CSF-induced membrane ruffling had lost the actin-cross-linking activity, and it inhibited the cross-linking activity of intact Iba1. These results indicate that Iba1 is a macrophage/microglia-specific actin-cross-linking protein essential for M-CSF-induced membrane ruffling.     

ATRA-regulated Asb-2 gene induced in differentiation of HL-60 leukemia cells

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C-terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N-terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL-60 cells, and demonstrated that ASB-2, one of the ASB proteins, was rapidly induced by all-trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb-2 gene. We showed that RARalpha, one of the RARs, binds to the RARE/RXRE in the Asb-2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB-2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.     

CD39 modulates IL-1 release from activated endothelial cells

The activation of endothelial cells (EC) and monocyte-macrophages (Mφ) by lipopolysaccharide (LPS) is considered an important element of the vascular injury observed in endotoxemia. Interleukin-1 (IL-1) beta release from Mφ in response to LPS, appears to be mediated by the autocrine/paracrine release of ATP via P2X7 receptor activation. In EC, similar nucleotide-mediated signaling pathways may be influenced by high levels of expression of CD39, the vascular nucleoside triphosphate diphosphohydrolase (NTPDase; ENTPD I). To determine whether CD39 modulates ATP-mediated release of IL-1 from EC, we stimulated human EC with LPS and measured levels of ATP secretion and IL-1 release. LPS triggered ATP secretion from EC that was soon followed by IL-1alpha release. Overexpression of CD39 following infection with recombinant CD39 adenoviral vectors (AdCD39) abrogated the initial phase of ATP secretion and inhibited IL-1alpha release; comparable results were obtained with soluble NTPDase. These data demonstrate that CD39/NTPDase modulates IL-1alpha release from LPS stimulated human EC.