Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.     

Generation of rac3 null mutant mice: role of Rac3 in Bcr/Abl-caused lymphoblastic leukemia

Numerous studies indirectly implicate Rac GTPases in cancer. To investigate if Rac3 contributes to normal or malignant cell function, we generated rac3 null mutants through gene targeting. These mice were viable, fertile, and lacked an obvious external phenotype. This shows Rac3 function is dispensable for embryonic development. Bcr/Abl is a deregulated tyrosine kinase that causes chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia in humans. Vav1, a hematopoiesis-specific exchange factor for Rac, was constitutively tyrosine phosphorylated in primary lymphomas from Bcr/Abl P190 transgenic mice, suggesting inappropriate Rac activation. rac3 is expressed in these malignant hematopoietic cells. Using lysates from BCR/ABL transgenic mice that express or lack rac3, we detected the presence of activated Rac3 but not Rac1 or Rac2 in the malignant precursor B-lineage lymphoblasts. In addition, in female P190 BCR/ABL transgenic mice, lack of rac3 was associated with a longer average survival. These data are the first to directly show a stimulatory role for Rac in leukemia in vivo. Moreover, our data suggest that interference with Rac3 activity, for example, by using geranyl-geranyltransferase inhibitors, may provide a positive clinical benefit for patients with Ph-positive acute lymphoblastic leukemia.     

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M₃ muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M₃ mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca²⁺ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands. translocation; aquaporin-5     

Tau and GSK3β Dephosphorylations are Required for Regulating Pin1 Phosphorylation

Pin1 binds mitotically phosphorylated Thr231–Pro232 and Thr212–Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3β phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Aβ-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-β APP accumulation than C83-βAPP in APPsw-tansfectant and thereby promoted Aβ-42 production in transfectants. (ii) the inhibition of tau and GSK3β phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3β for protecting against Alzheimer’s disease.     

Characterization of the T-cell receptor gamma locus and analysis of the variable gene segment expression in rabbit

The genomic organization and expression of genes of the T-cell receptor gamma (TRG) locus are described for mice and humans, but not for species such as rabbits (Oryctolagus cuniculus), in which T cells compose a sizeable proportion of T cells in the periphery. We cloned 200 kb of the rabbit TRG locus and determined the TRGV gene usage in adult and newborn rabbits by RT-PCR. We identified two TRGJ genes, one TRGC gene, and 22 TRGV genes, all of which encoded functional variable regions. One TRGV gene is the unique member of the TRGV2 subgroup, whereas the other genes belong to the TRGV1 subgroup. Evolutionary analyses of TRGV1 genes identified three distinct groups that can be explained by separate duplication events in the rabbit genome. Evidence of gene conversion between TRGV1.1 and TRGV1.6 was observed. Both TRGV1 and TRGV2 subgroup genes were expressed in the spleen, intestine, and appendix of adult rabbits, and the repertoire of TRGV genes expressed in these tissues was similar. In these tissues from newborns, and in skin from adults, only the genes from the TRGV1 subgroup were expressed. Greater TRGV-J junctional diversity was found in tissues from adult compared to newborn rabbits. Our analyses indicate rabbits have a larger germ line encoded TRG repertoire compared with that of mice and humans. In addition, we found TRGV gene usage is alike in most tissues of rabbits similar to that found in humans but in contrast to that found in mice.Electronic SupplementaryMaterial Supplementary material is available for this article at The nucleotide sequence data reported in this article have been submitted to GenBank and are assigned the accession numbers AY748325–AY748348     

A partial-complementary adapter for an improved and simplified ligation-mediated suppression PCR technique

Ligation-mediated suppression PCR (LMS-PCR) is a powerful tool for walking in unknown genomic DNA regions from known adjacent sequences. This approach has made it feasible to obtain promoter sequences and to enable researchers to identify full-length gene sequences or isoforms of multigene families. However, the advantages of LMS-PCR can be obviated by the presence of incomplete base modifications on the suppression adapters. We propose here that a 'partial-complementary adapter' is a more reliable suppression adapter, demanding only 5'-end phosphorylation. We also describe a simplified procedure for the easier preparation of PCR templates with very small quantities of DNA and a fast and direct characterization of the suppression-PCR products. A set of practical guidelines is proposed for pre-checking the efficiency of the adapter modification using two model systems: bacteriophage lambda (lambda) and Arabidopsis.     

Identification and characterization of a putative cyclic nucleotide-gated channel, CNG-1, in C. elegans

Cyclic nucleotide-gated (CNG) channels encoded by the tax-4 and tax-2 genes are required for chemosensing and thermosensing in the nematode C. elegans. We identified a gene in the C. elegans genome, which we designated cng-1, that is highly homologous to tax-4. Partial CNG-1 protein tagged with green fluorescent protein was expressed in several sensory neurons of the amphid. We created a deletion mutant of cng-1, cng-1 (jh111), to investigate its in vivo function. The mutant worms had no detectable abnormalities in terms of their basic behavior or morphology. Whereas tax-4 and tax-2 mutants failed to respond to water-soluble or volatile chemical attractants, the cng-1 null mutant exhibited normal chemotaxis to such chemicals and a tax-4;cng-1 double mutant had a similar phenotype to tax-4 single mutants. Interestingly, cng-1 and tax-4 had a synergistic effect on brood size.     

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.