The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment.

Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incoming IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face.

Localization of polysome-bound albumin and serine dehydratase in rat liver cell fractions.

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [(125)I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [(125)I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [(125)I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5-6 times those released from free polysomes. Anti-rat serum albumin-[(125)I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [(125)I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[(125)I]Fab to free polysomes was also obtained. The [(125)I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[(125)I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.

CD4(+) Valpha14 natural killer T cells are essential for acceptance of rat islet xenografts in mice.

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. However, the precise cellular and molecular mechanisms of the immune reactions against allogeneic and xenogeneic transplanted islets remain unclear. Here, we demonstrate that CD4(+) Valpha14 natural killer T (NKT) cells, a recently identified lymphoid cell lineage, are required for the acceptance of intrahepatic rat islet xenografts. An anti-CD4 mAb, administrated after transplantation, allowed islet xenografts to be accepted by C57BL/6 mice, with no need for immunosuppressive drugs. The dose of anti-CD4 mAb was critical, and the beneficial effect appeared to be associated with the reappearance of CD4(+) NKT cells at around 14 days after transplantation. Interestingly, rat islet xenografts were rejected, despite the anti-CD4 mAb treatment, in Valpha14 NKT cell-deficient mice, which exhibit the normal complement of conventional lymphoid cells; adoptive transfer of Valpha14 NKT cells into Valpha14 NKT cell-deficient mice restored the acceptance of rat islet xenografts. In addition, rat islet xenografts were accepted by Valpha14 NKT mice having only Valpha14 NKT cells and no other lymphoid cells. These results indicate that Valpha14 NKT cells play a crucial role in the acceptance of rat islet xenografts in mice treated with anti-CD4 antibody, probably by serving as immunosuppressive regulatory cells.

Effects of interleukin-1 on syntheses of alkaline phosphatase, type X collagen, and 1,25-dihydroxyvitamin D3 receptor, and matrix calcification in rabbit chondrocyte cultures.

The effect of IL-1 on expression of the mineralization-related phenotype by chondrocytes was examined. In cultures of rabbit growth plate chondrocytes, IL-1 beta at 0.1 ng/ml caused 95% decreases in alkaline phosphatase activity, alkaline phosphatase mRNA levels, the incorporation of 45Ca into insoluble material, and the calcium content during the hypertrophic stage. These effects of IL-1 beta were dose-dependent and were observed in 24-48 h. Furthermore, IL-1 beta suppressed increase in cell size and the syntheses of 1,25-dihydroxyvitamin D3 receptor and type X collagen, other markers of hypertrophy, but had little effect on the synthesis of total protein including type II collagen. The inhibition of calcification was observed only when chondrocytes were exposed to IL-1 before the onset of calcification: IL-1 treatment from the mineralization stage had a marginal effect on 45Ca incorporation into insoluble material. These results suggest that IL-1 inhibits chondrocyte hypertrophy and the onset of calcification in ossifying cartilage.

The ectonucleotidases alkaline phosphatase and nucleoside triphosphate diphosphohydrolase 2 are associated with subsets of progenitor cell populations in the mouse embryonic, postnatal and adult neurogenic zones.

Subventricular zone (SVZ)-derived adult neurospheres express two ectonucleotidases, nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and tissue non-specific alkaline phosphatase (TNAP). Agonists of the nucleotide receptors P2Y(1) and P2Y(2) as well as adenosine augment growth factor-mediated progenitor cell proliferation. NTPDase2 converts ATP and UTP to ADP and UDP, respectively, which are all P2Y receptor agonists. TNAP hydrolyzes nucleoside triphosphates and diphosphates and produces the P1 receptor agonist adenosine. In the SVZ, NTPDase2 is specifically expressed by type B cells. In order to further scrutinize the association of key molecules of the purinergic signaling pathway with neurogenic regions, we analyzed the expression of TNAP at the lateral ventricles of the adult and developing mouse brain. In the adult brain, TNAP was expressed by type B, type A and at least subsets of type C cells of the SVZ and throughout the rostral migratory stream. Almost 100% of the proliferating, Ki-67-positive cells of the adult SVZ stained for TNAP, supporting the notion of a ubiquitous association of TNAP with SVZ progenitors. In contrast, NTPDase2-positive progenitors of the dentate gyrus were TNAP-negative. Essentially all cells of the telencephalic vesicle at embryonic day (E) 14 revealed TNAP activity, including doublecortin-positive neuroblasts. During further embryonic development, enhanced TNAP activity became restricted to cells of the ventricular and SVZ. In contrast to TNAP, NTPDase2 was first expressed in the SVZ perinatally, in association with TNAP-positive SVZ border cells. During later development, NTPDase2-positive cells disappeared from the ventricular surface and began to form sheaths around clusters of subventricular doublecortin-positive cells, apparently transforming into type B cells. Our results identify TNAP and NTPDase2 as novel markers for subsets of progenitors in the adult and developing mouse brain. They further support the notion that signaling via extracellular nucleotides and nucleosides contributes to embryonic and adult neurogenesis.

Elevation of alpha2-->6 sialyltransferase and alpha1-->2 fucosyltransferase activities in human choriocarcinoma.

Structures of N-linked sugar chains are species and tissue specific and change in the course of tumorigenesis. Sialyl linkages of human placental glycoproteins are exclusively Neu5Ac alpha2-->3Gal, whereas Fuc alpha1-->2Gal and Neu5Ac alpha2-->6Gal residues are expressed in human chorionic gonadotropin and alkaline phosphatase, which are produced in human choriocarcinoma JEG-3 and BeWo cells. In the present study, to elucidate the enzymological and molecular biological basis of the structural changes that occur in the course of tumorigenesis, alpha1-->2 fucosyltransferase, alpha2-->3 and alpha2-->6 sialyltransferase activities, and the expression levels of the corresponding mRNAs were measured. The alpha2-->3 sialyltransferase activity did not change as a result of tumorigenesis; however, the alpha2-->6 siayltransferase activity and alpha1-->2 fucosyltransferase activity in JEG-3 and BeWo cells increased to levels several times higher than those in placenta Competitive PCR analysis showed that the expression levels of mRNA encoding alpha1-->2 fucosyltransferase and mRNA encoding alpha2-->6 sialyltransferase increased significantly as a result of tumorigenesis, indicating that such structural changes are regulated at the level of transcription of these glycosyltransferase genes.

Lewis and secretor gene dosages affect CA19-9 and DU-PAN-2 serum levels in normal individuals and colorectal cancer patients.

The effect of doses of the secretor (Se) and Lewis (Le) genes on the serum levels of CA19-9 and DU-PAN-2 was investigated in 400 normal individuals. It was clearly demonstrated that the Se gene dosage negatively affected both the CA19-9 and DU-PAN-2 values, whereas the Le gene dosage positively affected the CA19-9 value and negatively affected the DU-PAN-2 value. The 400 normal individuals were separated into nine groups by their Le and Se genotypes, as follows: group 1, Le/Le and se/se; group 2, Le/le and se/se; group 3, Le/Le and Se/se; group 4, Le/le and Se/se; group 5, Le/Le and Se/Se; group 6, Le/le and Se/Se; group 7, le/le and se/se; group 8, le/le and Se/se; and group 9, le/le and Se/Se. The group 1 individuals, having homozygous inactive Se alleles (se/se) and homozygous active Le alleles (Le/Le), exhibited the highest mean CA19-9 value. The CA19-9 value clearly ranged from a high in group 1 to a low in group 9. All of the Le-negative individuals who had the le/le genotype (groups 7, 8, and 9) had completely negative CA19-9 values, i.e., under 1.0 unit/ml, irrespective of the Se genotype. Group 7 individuals (le/le and se/se) showed a higher mean DU-PAN-2 value than did individuals in other groups. The Le-negative individuals in groups 8 and 9 also showed a higher mean DU-PAN-2 value than did the Le-positive individuals in groups 1-6. We recommend that the revised Le and Se genotype-dependent positive/negative cutoff values for CA19-9 and DU-PAN-2, determined in this study, be applied for more accurate cancer diagnoses. The Le and Se genotypes of 168 patients with colorectal cancer were also examined, and the CA19-9 and DU-PAN-2 values were measured before surgical resection. All 15 Le-negative patients (le/le) with colorectal cancer again showed undetectable CA19-9 values, i.e., under 1.0 unit/ml, but many of them exhibited highly positive DU-PAN-2 values. In contrast, many of the Le-positive patients (Le/Le or Le/le) had positive CA19-9 values, whereas very few of them exhibited positive DU-PAN-2 values. CA19-9 measurement is more useful than is DU-PAN-2 measurement for Le-positive patients, but it is not useful for Le-negative ones. DU-PAN-2 measurement should be performed in Le-negative patients for cancer diagnosis.

Eradication diminishes enhancing effects of Helicobacter pylori infection on glandular stomach carcinogenesis in Mongolian gerbils.

To investigate the nature of the link between Helicobacter pylori (Hp) infection and stomach carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. MGs were treated with N-methyl-N-nitrosourea (MNU), followed by inoculation with Hp (groups 1 and 2) or without Hp (group 3), or infected with Hp (groups 4 and 5) or inoculation without Hp (group 6) followed by MNU administration. At week 21, the animals in groups 2 and 5 underwent an eradication procedure. At week 50, the incidences of adenocarcinomas in group 1 (15 of 23) and group 4 (9 of 26) were significantly higher than in group 3 (1 of 15) and group 6 (1 of 18), respectively. Moreover, those in group 2 (5 of 24) and group 5 (2 of 22) were lower than in groups 1 and 4, respectively. This study shows that Hp eradication may be useful as a prevention approach against stomach cancer.

Inhibitory effect of colchicine on translocation of alkaline phosphatase to the plasma membrane concomitant to its induction in rat liver.

A single injection of colchicine (1--3 mg/kg body weight) caused a remarkable induction of hepatic alkaline phosphatase, which increased linearly in the homogenate starting at 5--6 h and reached a maximum level (14-fold of the control activity) at 20--22 h after the drug treatment. In the plasma membrane, however, the increase in specific activity and the recovery of alkaline phosphatase were greatly inhibited up to 12 h after the treatment. Such an inhibitory effect of colchicine was confirmed by a combination experiment of the drug treatment with bile duct ligation; in the plasma membrane elevation of the enzyme induced by bile duct ligation was also greatly retarded by colchicine. The subcellular distribution of the enzyme activity in livers was determined among the four groups of rats with or without bile duct ligation and/or colchicine administration taken at 8 h after each treatment. In the control and the bile duct-ligated livers, the highest specific activity was observed in the plasma membrane fraction, while in the colchicine-treated livers, with or without bile duct ligation, the highest activity was found in the Golgi fractions. These results indicate that the Golgi membranes enriched with the induced enzyme were blocked by the drug to prevent migration toward the plasma membranes enriched with the induced enzyme were blocked by the drug to prevent migration toward the plasma membrane, thus demonstrating involvement of the Golgi complex in the translocation route of newly synthesized alklaine phosphatase to the plasma membrane.

Multiple forms of albumin and their conversion from pro-type to serum-type in rat liver in vivo.

Purified rat serum albumin was resolved into five forms by polyacrylamide gel isoelectric focusing, while albumin isolated from the subcellular fractions of rat liver consisted of those of serum albumin and four additional forms. This latter four forms were identified as proalbumin, since they were converted to those of serum albumin by limited proteolysis with trypsin. This method was applied to the determination of the conversion site of proalbumin in the liver, revealing that substantial conversion of proalbumin occurs in the Golgi cisternae as well as in the secretory vesicles.

Isolation of Golgi fractions from colchicine-treated rat liver. II. Electrophoretic characterization.

1. Intact Golgi fractions, three from colchicine- or ethanol-treated rat livers and two from a control, were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. All the fractions showed very similar electrophoretic profiles with 33 protein bands, some of which, especially albumin, had rather higher density in the secretory vesicle fraction than those in the cisternal fraction. 2. Using albumin as the content marker, the Golgi fractions were subfractionated into membranes and contents by freezing-thawing and sonication followed by centrifugation. Distribution of galactosyltransferase among these membrane preparations showed that this enzyme was more enriched in the Golgi cisternal membranes than in the secretory vesicle membranes. 3. All the membrane preparations from the Golgi complex showed very similar patterns on electrophoresis, which were distinctly different from those of microsomal membranes and of plasma membrane. Furthermore, all the Golgi content subfractions had similar protein components, most of which were also found in serum. The microsomal contents, however, showed a considerably different pattern from those of the Golgi contents. 4. From these results it could be concluded that the secretory vesicles are indeed a member of the Golgi complex despite their different appearance and morphology.

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).

Isolation of Golgi fractions from colchicine-treated rat liver. I. Morphological and enzymic characterization.

1. Three Golgi fractions, GF-1, GF-2 and GF-3, were isolated from the livers of rats pretreated with colchicine, which gave better yields of the fractions than ethanol treatment of rats. 2. Electron microscopic observation showed that GF-1 was composed mainly of secretory vesicles, GF-3 consisted predominantly of small tubules and flattened cisternae, and GF-2 was an intermediate fraction composed of secretory vesicles and cisternal elements. 3. Among these three fractions the highest activity of galactosyl transferase, marker enzyme of the Golgi complex, was found in GF-3 and the lowest activity was in GF-1, although a different distribution of the enzymes was observed in fractions obtained from ethanol-treated rat liver. 4. Enzymatic characterization of these fractions showed that no significant contamination with other subcellular components occurred in GF-1 and GF-2.

Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glu----Lys). A novel apolipoprotein variant.

A novel genetic variant of apolipoprotein(apo) A-I Fukuoka, has been identified in a Japanese family. This variant has a relative charge of +2 compared to normal apolipoprotein A-I (A-I4), on the isoelectric focusing gels and the same molecular mass and immunologic characteristics as normal apolipoprotein A-I. This variant, transmitted as an autosomal co-dominant inheritance was purified by preparative Immobiline isoelectric focusing. Sequence analysis after cleavage with lysyl endopeptidase and CNBr, followed by high-performance liquid chromatography revealed a single substitution of lysine at position 110, instead of the usual glutamic acid. This mutant apolipoprotein A-I has much the same potential as to activate lecithin-cholesterol acyltransferase.

Contrast manifestation of alkaline phosphatase and 5'-nucleotidase in plasma membranes isolated from rat liver and ascites hepatoma.

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.

The yeast SFL2 gene may be necessary for mating-type control.

We previously reported the isolation of the yeast suppressor gene for flocculation, SFL2 (TUP1). SFL2 gene disruption results in pleiotropic phenotypes; the sfl2 null mutation also causes a morphological change similar to shmoo in both the MAT alpha and MATa/alpha cells. The MAT alpha and MATa/alpha sfl2 null mutant cells incorporate chitin into the new growth zone in the same way as the alpha-factor-treated MATa cells. In order to clarify the molecular basis of this morphological change, we examined the effect of the sfl2 null mutation on the mRNA production of various genes involved in mating-type control. The transcripts of both the STE2 (an a-specific gene) and STE3 (an alpha specific gene) genes are detected in the MAT alpha and MATa/alpha cells carrying the sfl2 null mutation. In addition, mRNA of the GPA1 gene (haploid-cell-specific gene) is also detected in the MATa/alpha sfl2 cells. However, there is no significant difference in the levels of the MAT alpha 2 and MATa1 transcripts. These results suggest that the SFL2 gene product may be necessary for alpha 2 and a1-alpha 2 repression.

Biosynthesis and processing of pro-C3, a precursor of the third component of complement in rat hepatocytes: effect of secretion-blocking agents.

Biosynthesis and intracellular processing of the third component (C3) of complement were studied in cultured rat hepatocytes. In the control cells, the complement C3 was synthesized as a pro-form, a single polypeptide chain comprising both the alpha- and beta-subunits. Although the cleavage of the pro-form into the subunits was not clearly demonstrable within the cells during pulse-chase periods, all the secreted C3 was the mature processed form. The cells were treated with secretion-blocking agents with different modes of action, colchicine and monensin. Colchicine caused an accumulation of the processed C3 within the cells, whereas monensin blocked the secretion without a significant accumulation of the processed form. The results indicate that the conversion of the C3 pro-form into the subunits takes place in the secretory vesicles just before the secretion.

Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes.

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a precursor (pro-C3), which was processed to the mature form with alpha- and beta-subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro-C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro-C3 to the Golgi complex where pro-C3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.

Alpha1,3-fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX.

The amino acid sequence of Fuc-TIX is very highly conserved between mouse and human. The number of non-synonymous nucleotide substitutions of the Fuc-TIX gene between human and mouse was strikingly low, and almost equivalent to that of the alpha-actin gene. This indicates that Fuc-TIX is under a strong selective pressure of preservation during evolution. The human Fuc-TIX (hFuc-TIX) showed a unique characteristics, i.e. hFuc-TIX was not activated by Mn2+ and Co2+, whereas hFuc-TIV and hFuc-TVI were activated by the cations. The hFuc-TIX transcripts were abundantly expressed in brain and stomach, and interestingly were detected in spleen and peripheral blood leukocytes.

A novel glycosyltransferase with a polyglutamine repeat; a new candidate for GD1alpha synthase (ST6GalNAc V)(1).

The fifth type GalNAcalpha2,6-sialyltransferase (mST6GalNAc V) was cloned from a mouse brain cDNA library. mST6GalNAc V exhibited type II transmembrane topology containing a polyglutamine repeat, which showed 42.6% and 44.8% identity to mouse ST6GalNAc III and IV, respectively. Northern blot analysis revealed that the mST6GalNAc V gene was specifically expressed in forebrain and cerebellum. mST6GalNAc V exhibited GD1alpha synthetic activity from GM1b the same as mST6GalNAc III and IV. The activity ratio of GM1b toward fetuin and the expression pattern were completely different among the three ST6GalNAcs. Interestingly, the polyglutamine repeat number was different from that of inbred mice. We report the first glycosyltransferase with a polymorphic polyglutamine repeat.