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A study of AXL1 gene present in a mutant yeast reveals that it is a haploid cell which produces a protein similar to insulin degrading enzymes in Drosophila and human beings and carries out a budding pattern different from that of a/alpha diploid cells. The ectopic expression of AXL1 resulted in an axial budding pattern in a/alpha cells, suggesting that Axl1 helps to convert bipolar budding to axial. The findings also show that Bud3 and Bud4 play significant roles in promoting axial budding with Axl1.

A study was conducted to determine the basis of dipeptidyl peptidase IV (DPPIV) activity deficiency in the Fischer rat substrain 344/CRJ at the molecular level. Through northern blot analysis, it was found that normal and 344/CRJ rats both had the same size and amount of DPPIV mRNA. DPPIV cDNA cloning and sequencing, however, found that a G to A transition occurred at nucleotide 1897 in 344/CRJ's cDNA sequence. This caused a substitution of Gly633 -> Arg in the active site sequence Gly629-Trp-Ser-Tyr-Gly633. It was found that a single Gly633 -> Arg substitution is enough to induce rapid intracellular degradation in the mutant DPPIV in the endoplasmic reticulum.

Chemical alteration and site-directed mutagenesis were used to study the catalytic site of dipeptidyl peptidase IV (DPPIV). High performance liquid chromatographic and sequence analyses of peptide fragments from labeled DPPIV reveal that the label was conjugated at a serine residue in a sequence corresponding to serine proteases consensus sequence. Site-directed mutation of the other residues in the sequence show that it is necessary for normal enzyme activity.

Tumor necrosis factor-α (TNFα)-induced reactions are effective to maintain homeostasis; however, excessive responses play progressive roles in the pathogenesis of various chronic inflammatory diseases. We demonstrate that TNFα triggered the release of its receptor TNFR1 as a content of extracellular vesicles (EVs) from the human bronchial epithelial cell, BEAS-2b. The TNFR1 cytoplasmic domain binding partner, TNFR-associated death domain (TRADD), was released by TNFα treatment along with TNFR1. TNFα-triggered release of EVs was decreased in the presence of amitriptyline, an inhibitor of acid sphingomyelinase (A-SMase), or of GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), indicating that EVs containing TNFR1 and TRADD are released through A-SMase and N-SMase dependent manners. From sucrose density gradient analysis, each sphingomyelinase is involved in the generation of distinct populations of EVs. Inhibition of A-SMase or N-SMase resulted in significantly increased responses to TNFα in parental cells. Given that TRADD serves as a platform for the assembly of subsequent signaling molecules, the TNFα triggered release of TNFR1 and TRADD might be an effective strategy for down regulation of the TNFα responses of parental cells. [ABSTRACT FROM AUTHOR]

p230/golgin-245 is a trans -Golgi coiled-coil protein that is known to participate in regulatory transport from the trans -Golgi network (TGN) to the cell surface. We investigated the role of p230 and its interacting protein, microtubule actin crosslinking protein 1 (MACF1), in amino acid starvation-induced membrane transport. p230 or MACF1 knock-down (KD) cells failed to increase the autophagic flow rate and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive puncta under starvation conditions. Loss of p230 or MACF1 impaired mAtg9 recruitment to peripheral phagophores from the TGN, which was observed in the early step of autophagosome formation. Overexpression of the p230-binding domain of MACF1 resulted in the inhibition of mAtg9 trafficking in starvation conditions as in p230-KD or MACF1-KD cells. These results indicate that p230 and MACF1 cooperatively play an important role in the formation of phagophore through starvation-induced transport of mAtg9-containing membranes from the TGN. In addition, p230 itself was detected in autophagosomes/autolysosome with p62 or LC3 during autophagosome biogenesis. Thus, p230 is an important molecule in phagophore formation, although it remains unclear whether p230 has any role in late steps of autophagy. [ABSTRACT FROM AUTHOR]

Abstract: Strains of Thermus thermophilus produce unique carotenoids called thermozeaxanthins and their colonies are light-yellow pigmented. Here, we developed a new cloning system allowing for the rapid and convenient detection of recombinants by color screening based on carotenoid production in T. thermophilus. We constructed two cloning vectors that overexpress the crtB gene encoding a phytoene synthase under the strong promoter of the slpA gene. Phytoene synthase is one of essential enzymes for the production of carotenoids. We also isolated a carotenoid-overproducing mutant that formed orange colonies. Because disruption of crtB in the carotenoid-overproducing mutant resulted in white colonies, we used the disruptant as a host strain. Whereas transformants carrying a new cloning vector, pTRK1-PRslpA-crtBcas, grew into unusual red-pigmented colonies probably because of the extreme accumulation of thermozeaxanthins, those carrying the vector with a foreign DNA inserts formed white colonies. Thus, recombinants can be detected easily by color screening (red/white screening) in T. thermophilus. This cloning system requires no additional chromogenic substrate in the medium. We also constructed a promoter-probe vector, pTRK1-crtBmcs-PP, employing the open reading frame of crtB with multiple cloning sites. Using this vector, a series of colony-color phenotypes is observed probably depending on promoter activities of foreign DNA inserts, which enables the rapid probing of promoters. These vectors are useful to simplify cloning procedures and to identify the promoters of different strengths in T. thermophilus. [Copyright &y& Elsevier]

Background: Dravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Nav1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome's human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs. Results: We generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected. Conclusions: Our results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies. [ABSTRACT FROM AUTHOR]

Abstract: Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZα gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus. [Copyright &y& Elsevier]

In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from monopolar to bipolar in character, which is known as ‘new end take off’ (NETO). We previously found that arf6p, a member (class III) of the ADP-ribosylation factor (Arf) family, is necessary for NETO in fission yeast. Here we report the characterization of an S. pombe gene, syt22+, encoding a putative Arf guanine nucleotide exchange factor (GEF). The syt22 protein contains a Sec7 domain and a PH domain conserved in the mammalian EFA6 GEF family, and has high similarity to Yel1p, which was identified as a GEF for Arf3p (class III Arf) in Saccharomyces cerevisiae. syt22Δ cells, like arf6Δ cells, completely failed to undergo NETO. Syt22p uniformly localizes to the cell periphery. Its localization is not dependent on microtubules, actin cytoskeletons or arf6p. We hypothesize that syt22p functions as a GEF for arf6p. [ABSTRACT FROM AUTHOR]

Abstract: Depletion of p115 with small interfering RNA caused fragmentation of the Golgi apparatus, resulting in dispersed distribution of stacked short cisternae and a vesicular structure (mini-stacked Golgi). The mini-stacked Golgi with cis- and trans-organization is functional in protein transport and glycosylation, although secretion is considerably retarded in p115 knockdown cells. The fragmented Golgi was further disrupted by treatment with breferdin A and reassembled into the mini-stacked Golgi by removal of the drug, as observed in control cells. In addition, p115 knockdown cells maintained retrograde transport from the Golgi to the endoplasmic reticulum, although the rate was not as efficient as in control cells. While no alternation of microtubule networks was found in p115 knockdown cells, the fragmented Golgi resembled those in cells treated with anti-microtubule drugs. The results suggest that p115 is involved in vesicular transport between endoplasmic reticulum and the Golgi, along with microtubule networks. [Copyright &y& Elsevier]

GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH[sub 2]terminal region (positions 137-237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [³H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys[sup 69] and Cys[sup 72], accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface. [ABSTRACT FROM AUTHOR]

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, blocks protein transport from the endoplasmic reticulum (ER) to the Golgi complex and induces the redistribution of Golgi proteins into the ER. We investigated characteristics of NDGA-induced retrograde movement of the Golgi proteins to the ER. At an early stage of incubation of cells with NDGA, the Golgi complex formed convoluted membrane aggregates. Electron microscopy revealed that these aggregates directly interact en bloc with the ER membrane. The direct interaction and subsequent incorporation of the Golgi proteins into the ER were found to be temperature-dependent. The protein of ER–Golgi intermediate compartment (ERGIC), ERGIC53, was rapidly accumulated in the Golgi upon treatment with NDGA. This accumulation was significantly inhibited by low temperature at 15 °C. Under the condition, the redistribution of the Golgi proteins into the ER as well as the direct interaction between the ER and the Golgi by NDGA were also inhibited, suggesting an important role of the ERGIC in the retrograde movement. In contrast, the low temperature did not inhibit formation of the Golgi aggregates by NDGA. Taken together, these results suggest that NDGA causes the redistribution of the Golgi proteins into the ER through the direct connections between the Golgi, the ERGIC, and the ER. [Copyright &y& Elsevier]

The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast twohybrid screening identified GABA RAP (GABAA receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit. [ABSTRACT FROM AUTHOR]

A cDNA was cloned coding for human placental 5’-nucleotidase. The 3547-cDNA contains an open reading frame that encodes a 574-residue polypeptide with a calculated size of 63375 Da. The NH2-terminal 26 residues comprises a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. Four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophosholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5’-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 540-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the nmature 5’-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5’-nucleotidase. [ABSTRACT FROM AUTHOR]

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [³H]mannose,[³H]galactose, or [³H]fucose, all the radioactive precursors were incorporated into the β subunit of haptoglobin. [³]HMannose-labled haptoglobin was purified from the culture medium by immunoaffinity chromatogrpahy, and [³H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavali-A - Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [³H]fucose or [³H]galactose were also characterized by the above methods. The results indicated that rat haptoglobin contains two complex-type oligosccharide chains in each β subunit one with a possible structure of (± NeuAc→Galβ→GlcNAcβ→)3(Manα→)2Manβ→GlcNAc→(±Fucα→)GcNAc and the other with (±NeuAc→Galβ→GlcNacβ→Manα→)2Manβ→GlcNAc→(±Fucα→)GlcNAc. [ABSTRACT FROM AUTHOR]

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, α1-protease inhibitor and α2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, α1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51000, and then processed to two endoglycosidase-H-resistant forms having Mr 51000 and 56000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for α2u-globulin. In the cells lrcated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed α1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized α1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant α1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins. [ABSTRACT FROM AUTHOR]

The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A) RNA into polysomes was studied in isolated cells of Xenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A) RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached. The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A) RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%-25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis. [ABSTRACT FROM AUTHOR]

We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses (∼80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218↑Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162↑Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphospha-tidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162→Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation. [ABSTRACT FROM AUTHOR]

Mouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and VK) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and VK genes to the human heavy and light chain constant region genes (Cγ1 and CK) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches. [ABSTRACT FROM AUTHOR]

A cDNA encoding a 5′-nucleotidase was identified by screening a λgt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino adds, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5′-nucleotidase shares 61 % amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5′-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5′-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5–7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5′-nucleotidase, 3′-nucleotidase or phosphodiesterase activity. 5′-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5′-nucleotidase from multifunctional nucleotide hydrolases is discussed. [ABSTRACT FROM AUTHOR]

Structural analysis of 55 nearly full-length cDNA clones revealed heterogeneity in the 5'-untranslated regions of rat liver alkaline phosphatase mRNAs. The 5' extremities diverged into two totally unrelated sequence stretches at the position 88 nucleotides upstream of the initiation codon ATG. These two sequences, referred to as E1 and E2, were assigned on the genome about 36 000 base pairs (36 kbp) and 10 kbp upstream, respectively, of the exon coding for the 5'-most part of the common region. The gene consisted of 13 exons, including E1 and E2, and spanned about 56 kbp. The 11 exons (E3 to E13) following E1 and E2 were shared in common by the El-type and the E2-type mRNAs. Analyses by SI nuclease mapping and primer extension revealed the presence of two independent transcription-initiation sites specific to each of the El and E2 sequences. These results are interpreted as indicating a possible alternative usage of two leader exons, hence the presence of two independent promoters. Structural features of these putative promoters are described in the context of transcriptional fundamental and regulatory cis-elements. [ABSTRACT FROM AUTHOR]