Glycosylation site for chondroitin sulfate on the neural part-time proteoglycan, neuroglycan C

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate (CS) proteoglycan that is expressed predominantly in the central nervous system (CNS). NGC dramatically changed its structure from a proteoglycan to a nonproteoglycan form with cerebellar development, whereas a small portion of NGC molecules existed in a nonproteoglycan form in the other areas of the mature CNS, suggesting that the CS glycosylation of NGC is developmentally regulated in the whole CNS. As primary cultured neurons and astrocytes from cerebral cortices expressed NGC in a proteoglycan form and in a nonproteoglycan form, respectively, CS glycosylation seems to be regulated differently depending on cell type. To investigate the glycosylation process, cell lines expressing a proteoglycan form of NGC would be favorable experimental models. When a mouse NGC cDNA was transfected into COS 1, PC12D, and Neuro 2a cells, only Neuro 2a cells, a mouse neuroblastoma cell line, expressed NGC bearing CS chains. In PC12D cells, although three intrinsic CS proteoglycans were detected, exogenously expressed NGC did not bear any short CS chains just like NGC in the mature cerebellum. This suggests that the addition of CS chains to the NGC core protein is regulated in a manner different from that of other CS proteoglycans. As the first step in investigating the CS glycosylation mechanism using Neuro 2a cells, we determined the CS attachment site as Ser-123 on the NGC core protein by site-directed mutagenesis. The CS glycosylation was not necessary for intracellular trafficking of NGC to the cell surface at least in Neuro 2a cells.     

Expansion of human T-cell leukemia virus type 1 (HTLV-1) reservoir in orally infected rats: inverse correlation with HTLV-1-specific cellular immune response

Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals. Although the critical risk factor for ATL development is not clear, it has been noted that ATL is incidentally associated with mother-to-child infection, elevated proviral loads, and weakness in HTLV-1-specific T-cell immune responses. In the present study, using a rat system, we investigated the relationships among the following conditions: primary HTLV-1 infection, a persistent HTLV-1 load, and host HTLV-1-specific immunity. We found that the persistent HTLV-1 load in orally infected rats was significantly greater than that in intraperitoneally infected rats. Even after inoculation with only 50 infected cells, a persistent viral load built up to considerable levels in some orally infected rats but not in intraperitoneally infected rats. In contrast, HTLV-1-specific cellular immune responses were markedly impaired in orally infected rats. As a result, a persistent viral load was inversely correlated with levels of virus-specific T-cell responses in these rats. Otherwise very weak HTLV-1-specific cellular immune responses in orally infected rats were markedly augmented after subcutaneous reimmunization with infected syngeneic rat cells. These findings suggest that HTLV-1-specific immune unresponsiveness associated with oral HTLV-1 infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, but could be overcome with immunological strategies.     

Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony-stimulating factor (M-CSF). Their apoptosis was associated with a rapid and sustained increase in the pro-apoptotic BH3-only Bcl-2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c-Cbl. Although the number of OCs was increased in the skeletal tissues of bim-/- mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim-/- animals showed a marked prolongation of survival in the absence of M-CSF, compared with bim+/+ OCs, but the bone-resorbing activity of bim-/- OCs was significantly reduced. Overexpression of a degradation-resistant lysine-free Bim mutant in bim-/- cells abrogated the anti-apoptotic effect of M-CSF, while wild-type Bim did not. These results demonstrate that ubiquitylation-dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs.     

Generation of a polyclonal antibody that simultaneously detects multiple Ser/Thr protein kinases

In order to obtain a polyclonal antibody that recognizes various protein kinases, a peptide corresponding to an amino acid sequence of a highly conserved subdomain (subdomain VIB) of the protein kinase family was synthesized and used for immunization. When the synthetic peptide, CVVHRDLKPENLLLAS, was coupled to keyhole limpet hemocyanin (KLH) and used to immunize rabbits, polyclonal antibodies that detected multiple protein kinases on a Western blot were generated. One of the antibodies obtained, KI98, detected a variety of purified Ser/Thr protein kinases, such as calmodulin-dependent protein kinase II (CaM-kinase II), calmodulin-dependent protein kinase IV (CaM-kinase IV), cAMP-dependent protein kinase, protein kinase C, and Erk2. The antibody detected as low as 0.2 ng of protein kinases blotted onto a nitrocellulose membrane by dot-immunobinding assay. When a rat brain extract was analyzed with this antibody, various protein kinases were simultaneously detected. The present anti-peptide antibody with a broad spectrum of cross-reactivity to multiple protein kinases may be a powerful tool for comprehensive analysis focused on protein kinases.     

Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.     

Cloning and mapping of cat (Felis catus) immunoglobulin and T-cell receptor genes

 Molecular cloning and chromosomal mapping of the cat immunoglobulin (Ig) and T-cell receptor (TcR) genes were carried out to provide basic information for genetic analysis of immunologic diseases including leukemias and lymphomas in cats. We cloned two Ig constant genes, IGHM and IGHG and three TcR constant genes, TRAC, TRGC, and TRDC, by polymerase chain reaction (PCR) amplification of cDNA from cat peripheral blood mononuclear cells. For chromosomal mapping of the Ig and TcR loci including the IGK, IGL, and TRB on the cat genome, we performed PCR screening of DNAs from 37 cat × rodent somatic cell hybrids by using specific primers for the given genes. Consequently, three loci for IGH, TRA, and TRD, and two loci for TRB and TRG were found to be syntenic and assigned to cat chromosomes (FCA) B3 and A2, respectively. Further, IGK and IGL loci were mapped on FCA A3 and D3, respectively. These findings support the notion that the genetic linkages between the Ig and TcR genes are extensively conserved between humans and cats. Received: 18 June 1997 / Revised: 12 August 1997     

Microbiological activities of nucleotide loop-modified analogues of vitamin B12

Novel vitamin B₁₂ analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B₁₂- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5.6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B₁₂ activity and behaved as weak B₁₂ antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. ON the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [³H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B₁₂ activity toward E. coli 215 by blocking the B₁₂-transport systemAbbreviations AdoCbl adenosylcobalamin - MeCbl methylcobalamin - CN-Cbl cyanocobalamin or vitamin B₁₂ - Cbl cobalamin - (CN, aq)Cbi cyanoaquacobinamide - MeCbi methylcobinamide - Cbi cobinamide - (CN, aq)Cbi-PMe cyanoaquacobinamide methyl phosphate - Cbi-PMe cobinamide methyl phosphate - DBI 5,6-dimethylbenzimidazole - DBIyl 5,6-dimethylbenzimidazolyl - FMNH₂ fully reduced form of riboflavin 5-phosphate     

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.     

Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells

The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.