Controlled semihydrogenation of aminoalkynes using ethylenediamine as a poison of Lindlar's catalyst.
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Journal Article↗
Biomedical subjects
R D Larsen
Publications and source records attributed to R D Larsen.
zeta-, epsilon-, and gamma-Globin mRNA in blood samples and CD71(+) cell fractions from fetuses and from pregnant and nonpregnant women, with special attention to identification of fetal erythroblasts.
BACKGROUND: Information about the appearance of gamma-, epsilon-, and zeta-globin mRNAs in fetal erythroblasts during gestation and about the presence and amounts of these mRNAs in pregnant and nonpregnant women is important from the perspective of using these molecules as a marker of fetal erythroblasts. A specific marker is necessary for isolation and identification of fetal nucleated red blood cells from maternal blood samples for use in antenatal diagnosis of fetal genetic or chromosomal abnormalities. METHODS: We used a very sensitive reverse transcription-PCR (RT-PCR) method, coamplification analysis of gamma- and epsilon-globin cDNA, and quantitative analysis of gamma-globin mRNA based on competitive RT-PCR to investigate these aspects. RESULTS: All adult whole-blood samples were negative for epsilon- and zeta-globin mRNA. Analyses of CD71(+) cell fractions showed that specimens from 19 of 20 nonpregnant and 10 of 14 pregnant women (at 9-13 weeks of gestation) were positive for gamma-globin mRNA (Fisher's exact test, P = 0.13), and those from 3 of 20 nonpregnant and 5 of 14 pregnant women were positive for zeta-globin mRNA (Fisher's exact test, P = 0.23). No epsilon-globin mRNA was detected in CD71(+) cell fractions from 1-mL blood samples from adults. CD71(+) cell fractions from eight fetal blood samples (at 17-20 weeks of gestation) were positive for all three globin mRNAs. We found no statistically significant difference between the amounts of gamma-globin mRNA in pregnant and nonpregnant women. CONCLUSIONS: This study indicates that epsilon-globin mRNA might function as a marker for fetal CD71(+) cells early in pregnancy. Although gamma-globin mRNA can be detected in CD71(+) cell fractions from most adults, these transcripts also may be of use because of a marked difference between adult and fetal values.
Adult↗
A practical synthesis of a COX-2-specific inhibitor.
A number of synthetic strategies to the Cox-2 specific inhibitor 1 have been described. These studies have led to the identification of a novel pyridine construction using annulation of ketone 2 using a vinamidinium species 29 and ammonia in 97% assay yield. Three approaches to the synthesis of ketone 2 are described that allow for its preparation in large quantities in >65% overall yield from methyl 6-methylnicotinate.
Anti-Inflammatory Agents, Non-Steroidal↗
Neural tube defects among Mexican Americans living on the US-Mexico border: effects of folic acid and dietary folate.
Populations of Mexican descent have high occurrences of neural tube defects (NTDs). A recent study suggested that folic acid supplements may not protect these populations from NTDs. In a case-control study, the authors investigated the role of folic acid and dietary folate intake in NTD risk among Mexican Americans living along the Texas-Mexico border. From January 1995 to February 1999, 148 Mexican-American women with NTD-affected pregnancies and 158 women with normal live births were interviewed in person about use of vitamin supplements and dietary intakes during a 6-month periconceptional period (from 3 months before conception to 3 months after conception). Daily preconceptional consumption of vitamin supplements containing folic acid was 2.5% in control women and 2.0% in case women (odds ratio = 0.77; 95% confidence interval (CI): 0.19, 3.22). With adjustment for maternal age, education, obesity, and previous stillbirth or miscarriage, the risk estimate was essentially null (odds ratio = 1.12; 95% CI: 0.22, 5.78). Combined folic acid intake from diet and supplements showed only a modest risk reduction for intakes of > or = 1.0 mg per day (adjusted odds ratio = 0.73; 95% CI: 0.31, 1.72). The fact that the primary folic acid exposure was in the form of dietary polyglutamates rather than the more easily absorbed supplemental monoglutamates may explain an apparent decreased effect in this population.
Adult↗
Practical modifications and applications of the sharpless asymmetric aminohydroxylation in the one-pot preparation of chiral oxazolidin-2-ones.
[reaction: see text] Chiral oxazolidin-2-ones are synthetically valuable as chiral auxiliaries, and many have pharmaceutically interesting biological activity. This communication focuses on a convenient, practical one-pot preparation of chiral 4,5-disubstituted oxazolidan-2-ones in good yield with high enantioselectivities, using a modified Sharpless asymmetric aminohydroxylation of beta-substituted styrene derivatives followed by base-mediated ring closure. This procedure has been demonstrated on both small and large scale, utilizing 1, 3-dichloro-5,5-dimethyl hydantoin as an easily handled, commercially available substitute for tert-butyl hypochlorite.
Journal Article↗
An efficient preparation of vinamidinium hexafluorophosphate salts.
Substituted acetic acids or acetyl chlorides react with phosphorus oxychloride in DMF to yield the vinamidinium salts 3a-j in moderate to excellent recrystallized yields (28-90%). The cations are conveniently isolated as their hexafluorophosphate salts, which are easily handled nonhygroscopic solids. The nitro compound 3l is prepared in 91% yield by nitration of the parent vinamidinium 3k. The X-ray crystal structure is reported for the 2-phenyl isomer 3e and displays minimal overlap of the two pi-systems.
Anti-Inflammatory Agents, Non-Steroidal↗
Annulation of ketones with vinamidinium hexafluorophosphate salts: an efficient preparation of trisubstituted pyridines.
alpha-Aryl ketones react with vinamidinium hexafluorophosphate salts to give access to the corresponding 3-arylpyridines. The annulation reactions proceed in good to excellent yields with vinamidinium salts containing electron-withdrawing groups at the beta-position (R(2)). The reaction was applied to the preparation of the COX-2 specific inhibitor 5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine (1), as well as a series of analogues.
Cyclooxygenase Inhibitors↗
Neural tube defects along the Texas-Mexico border, 1993-1995.
In response to a 1991 anencephaly cluster in Cameron County, Texas, a surveillance and neural tube defect (NTD) recurrence prevention project for NTDs was implemented in the 14 Texas-Mexico border counties. For 1993-1995, NTD-affected pregnancies were identified at all gestational ages through active surveillance of multiple case-ascertainment sources. There were 87 cases of anencephaly, 96 cases of spina bifida, and 14 cases of encephalocele for respective rates of 6.4, 7.1, and 1.1 per 10,000 live births. Of the 197 NTD case-women, 93% were Hispanic. The overall, Hispanic, and Anglo NTD rates were, respectively, 14.6, 14.9, and 10.6 per 10,000 live births. The NTD rate for El Paso County (9.8 per 10,000), the most northwestern Texas county, was significantly lower (p = 0.001) than the aggregate rate for the rest of the Texas border (17.1 per 10,000). The overall Texas border rate was significantly higher (p < 0.001) than a recently estimated rate of 9.3 for California and minimally higher than a recently adjusted rate of 11.3 for the Metropolitan Atlanta Congenital Defects Program counties (p = 0.052), both of which now reflect all gestational ages. Of the 197 Texas border cases, 85% (168 cases) reached a gestational age of > or =20 weeks. Excluding cases of <20 weeks' gestation in the rate had a more marked effect on reducing the anencephaly rate (4.9 per 10,000) than the spina bifida rate (6.7 per 10,000). A country of birth was known for 153 (83%) of the 184 Hispanic case-women: 63% were born in Mexico; 24%, in Texas; and 11%, elsewhere in the United States. Rates for Mexico-born Hispanic women (15.1 per 10,000) were significantly higher than rates for United States-born Hispanic women (9.5 per 10,000) (p = 0.006).
Adult↗
Expression of human H-type alpha1,2-fucosyltransferase encoding for blood group H(O) antigen in Chinese hamster ovary cells. Evidence for preferential fucosylation and truncation of polylactosamine sequences.
The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type alpha1, 2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type alpha1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.
ABO Blood-Group System↗
Molecular cloning and characterization of CFT1, a developmentally regulated avian alpha(1,3)-fucosyltransferase gene.
Although coordinate expression of carbohydrate epitopes during development is well described, mechanisms which regulate this expression remain largely unknown. In this study we demonstrate that developing chicken B cells express the LewisX terminal oligosaccharide structure in a stage-specific manner. To examine regulation of this expression, we have cloned and expressed the chicken alpha(1,3)-fucosyltransferase gene involved in LewisX biosynthesis, naming it chicken fucosyltransferase 1 (CFT1). CFT1 is characterized by a single long open reading frame of 356 amino acids encoding a type II transmembrane glycoprotein. The domain structure and predicted amino acid sequence are highly conserved between CFT1 and mammalian FucTIV genes (52.8% and 46.3% identity to mouse and human respectively). In vitro CFT1 fucosyltransferase activity utilizes LacNAc > 3'sialyl-LacNAc acceptors with almost no utilization of other neutral type II (lactose, 2-fucosyllactose), or type I (lacto-N-biose I) acceptors. CFT1-transfected cells make cell surface LewisX (COS-7) and LewisX + VIM-2 structures (Chinese hamster ovary). CFT1 gene expression is tissue-specific and includes embryonic thymus and bursa. Furthermore, expression of the CFT1 gene and cell surface LewisX structures are closely linked during B cell development. These findings reveal the evolutionary conservation between nonmammalian and mammalian alpha(1,3)-fucosyltransferase genes and demonstrate a role for fucosyltransferase gene regulation in the developmental expression of oligosaccharide structures.
Amino Acid Sequence↗
Oncogene-related serum proteins and cancer risk: a nested case-control study.
Proto-oncogenes are genes coding for factors involved in cellular growth, reproduction, and differentiation. Cancer results through mutations of proto-oncogenes or through other mechanisms involving the products of proto-oncogenes. This study asks whether serum proteins immunologically related to the products of proto-oncogenes distinguish older men and women who manifest a new cancer during a 2-year follow-up. The authors conducted a nested case-control study that involved 248 men and women selected from a larger group of older (age > or = 65 years) healthy volunteers in a randomized clinical trial of preventive clinical services. Study subjects included 37 with a fatal cancer, 59 non-fatal breast, prostate, colon, or lung cancer, 58 hospitalized with at least one discharge diagnosis that coded to benign neoplasia (International Classification of Diseases, 9th Revision codes 210-239), and 94 randomly selected controls. Using seven monoclonal antibodies prepared against ras, erb-B, FES, myb, and SIS polypeptide sequences, immunoblots detected 17 proteins in serum collected from subjects before the clinical recognition of cancer. Five oncogene-related serum proteins appeared to distinguish older persons who manifested fatal (but not non-fatal) cancer over a brief (2-year) follow-up. Older persons hospitalized with benign neoplasia also had higher levels of these serum proteins. Relative to the 94 control subjects, a 52,000 dalton SIS-related protein (odd ratio (OR) = 5.9, 95% confidence interval (CI) 1.4-24.9) and a 35,000 dalton k-ras-related protein (OR = 11.3, 95% CI 1.2-104) were particularly common in serum from the 37 subjects who manifested a fatal cancer.
Aged↗
Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc.
The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.
ABO Blood-Group System↗
Specificity analysis of three clonal and five non-clonal alpha 1,3-L-fucosyltransferases with sulfated, sialylated, or fucosylated synthetic carbohydrates as acceptors in relation to the assembly of 3'-sialyl-6'-sulfo Lewis x (the L-selectin ligand) and related complex structures.
Unique specificities of the cloned alpha 1,3-L-fucosyltransferases (FTs), FT III (Lewis type), FT IV (myeloid type), and FT V (plasma type), and the alpha 1,3-FTs of Colo 205 (colon carcinoma), HL 60 (myeloid), B142 (lymphoid), EKVX (lung carcinoma), and calf mesenteric lymph nodes (CMLN) were discerned with sulfated, sialylated, and/or fucosylated Gal beta 1,3/4GlcNAc beta-based acceptor moieties. (a) FT V was 1.0-, 20.8-, and 4.6-fold active in forming Lewis x, Lewis y, and 3'-alpha-galactosyl Lewis x, respectively. (b) FT III and FT V formed approximately 4-fold 3'-sulfo Lewis x, as compared to 3'-sialyl Lewis x. (c) FT IV showed great efficiency in forming 3'-sulfo Lewis x (249%) and Lewis x (345%) in mucin-type branched chains. (d) FT III, FT IV, and FT V formed 19%, 62%, and 47% 6-sulfo Lewis x as compared to Lewis x. (e) 6'-Sulfo Lewis x and 3'-sialyl-6'-sulfo Lewis x (GLYCAM ligand) were not synthesized from their immediate precursors by FT III, FT IV, or FT V. (f) FT III, FT IV, and FT V were 311%, 9%, and 188% active, respectively, with 2'-fucosyl lactose but were not active with 2'- fucosyl-6'-sulfo lactose. (g) FT III and FT V were 7.0- and 0.5-fold active in forming Lewis a as compared to Lewis x, whereas, FT IV was inactive. (h) FT III was -2.0-fold more active in forming 3'-alpha-galactosyl Lewis a than Lewis b. (i) FT III synthesized 6-sialyl Lewis a (40% efficiency as compared to Lewis a) from 6-sialyl type 1. (j) FT III did not act on 6'-sulfo or 6'-sialyl type 1 but was 106% and 22% active with 3'-sulfo and 6-sulfo type 1, respectively. (k) The Colo 205 FT activities with type 1 compounds almost paralleled that of FT III except for the low activity (9%) with Gal beta 1,3(NeuAc alpha 2, 6)GlcNAc beta-O-Bn, but with type 2 considerable differences between Colo 205 FT and FT III were noticed. (l) The alpha 1,3-FTs of CMLN, HL60, B142, and EKVX were 1.2-1.7 times active with Fuc alpha 1,2Gal beta 1,4GlcNAc beta- O-pNP and Gal alpha 1,3Gal beta 1,4 GlcNAc beta-O-Bn with respect to Gal beta 1,4GlcNAc beta-O-Al. (m) Both CMLN and HL60 FTs were 2-fold active with 3-sulfoGal beta 1,4GlcNAc in a mucin-type branch structure such as 3-sulfoGal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. (n) The 3'-sulfoLacNAc/acrylamide copolymer, either as an acceptor or as a competitive inhibitor, had the potential to distinguish myeloid type alpha 1,3-FT from the plasma type.
Animals↗
Expression of blood group Lewis b determinant from Lewis a: association of this novel alpha (1,2)-L-fucosylating activity with the Lewis type alpha (1,3/4)-L-fucosyltransferase.
Blood group H type 1 [Fuc alpha (1,2)Gal beta (1,3)GlcNAc beta-->] is known as the precursor structure of the blood group determinant, Lewis b [Fuc alpha (1,2)Gal beta (1,3)(Fuc alpha (1,4))GlcNAc beta-->]. Recently, a new biosynthetic route for Lewis b from Lewis a [Gal beta (1,3)(Fuc alpha (1,4))GlcNAc-->] was identified in human gastric carcinoma cells, colon carcinoma Colo 205, and ovarian tumor. The present study demonstrates the association of this new type of alpha (1,2)-L-fucosyltransferase (FT) activity with the Lewis-type alpha (1,3/4)-L-FT as follows: (i) the alpha (1,4)- and novel alpha (1,2)-FT activities of Colo 205 were much less inhibited than the alpha (1,3)-FT activity by N-ethylmaleimide [Ki(microM) = 714.0, 119.0, and 6.5 respectively]. (ii) The alpha (1,4)- and novel alpha (1,2)-FT activities emerged from a Sephacryl S-200 column in identical positions. (iii) A specific inhibitor (copolymer from 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-allyl and acrylamide) of alpha(1,4)-FT activity inhibited both alpha(1,4)- and alpha(1,2)-FT activities in Sephacryl S-200 column effluent to almost the same extent (approximately 80%); (iv) separation of the Lewis-type alpha(1,3/4)-FT from the plasma-type alpha(1,3)-FT by specific elution of the affinity column (bovine IgG glycopep-Sepharose) with lactose and further purification on a Sephacryl S-100 HR column showed that (a) the alpha(1,3)-FT activity was the inherent capacity of the Lewis-type FT (Colo 205 fraction L) since approximately 90% of both the alpha(1,4)- and alpha(1,3)-FT activities is inhibited by the copolymer, (b) the unique ability of catalyzing the alpha(1,2)-L-fucosylation of Gal in Lewis a structure and also the alpha(1,3)-L-fucosylation of Glc in lactose-based structure belonged to the Lewis type enzyme (Colo 205 fraction L), (c) a measurement of the [14C]fucosyl products arising from the two acceptors Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn and 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A1 (specific for alpha(1,2) and alpha(1,4), respectively) taken in the same incubation mixture showed mutual inhibition by the acceptors ([Km for the alpha(1,4)-specific acceptor, 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A], increased from 32 to 50 microM in the presence of 7.5 mM Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn, whereas Ki for the mutual inhibition of alpha(1,2)-FT activity by the former was 102 microM], and (d) the Lewis-type FT, in contrast to the plasma type FT, was highly effective in fucosylating complex glycopeptides. (iv) A cloned FT (FT III:Lewis type) and the Colo 205 Lewis-type FT (fraction L) showed similar activities toward various acceptors; the enzymatic product resulting from the action of cloned FT on Galbeta(1,3)(Fucalpha(1,4))GlcNAc-beta-O-Bn was identified by FAB mass spectrometry as the difucosyl compound. (v) An examination of six human cell lines indicated that the novel alpha(1,2)-FT activity associates with the alpha(1,4)-FT activity.
Autoradiography↗
Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group T-hapten.
The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Alpha 1,4galactosyltransferase activity and Gb3Cer expression in human leukaemia/lymphoma cell lines.
We have used two methods to evaluate the level of expression of Gb3Cer in several human leukaemia/lymphoma cell lines representative of the myeloid (K562, KG-1, HL-60, and lymphoid (Reh, Daudi, Raji, RPMI 8226, CCRF-CEM, MOLT-4) lineages blocked at varied stages of differentiation. TLC immunostaining of glycolipid extracts with a monoclonal antibody, 12-101, and FACS analysis with the same antibody were used to demonstrate that the expression of Gb3Cer in neoplastic myeloid and lymphoid cells is both lineage and differentiation dependent. As a possible control point in the regulated expression of Gb3Cer we have investigated the first committed step in the synthesis of globo series glycosphingolipids that involves UDP-Gal:LacCer alpha (1,4)-galactosyltransferase (alpha 1,4GalT). We present the first characterization of this enzyme in a human myeloid cell line using an ELISA-based assay, which was subsequently used to measure alpha 1,4GalT activity in the human leukaemia/lymphoma cell lines. In general, there is a positive correlation between the levels of endogenous Gb3Cer and the level of the alpha 1,4 GalT activity. However, in two cases (KG-1 and CCRF-CEM) the level of enzyme activity did not correspond to the level of Gb3Cer expression.
Carbohydrate Conformation↗
Chicken B cells undergo discrete developmental changes in surface carbohydrate structure that appear to play a role in directing lymphocyte migration during embryogenesis.
The migration of progenitor cells to specific microenvironments is essential for the development of complex organisms. Avian species possess a unique primary lymphoid organ, the bursa of Fabricius, that plays a central role in the development of B cells. B cell progenitors, however, arise outside the bursa of Fabricius and, during embryonic development, must migrate through the vasculature to the bursa of Fabricius. In this report, we demonstrate that these progenitor B cells express the sialyl Lewis x carbohydrate structure previously shown to be a ligand for the selectin family of vascular adhesion receptors. Soon after migration to the bursa of Fabricius, B cell progenitors are induced to undergo a developmental switch and terminate the expression of sialyl Lewis x in a temporal pattern that correlates with the developmental decline in the ability of these cells to home to the bursa of Fabricius upon transplantation. The induction of the developmental switch in the glycosylation pattern of developing B cells requires the bursal environment. In addition, sialyl Lewis x carbohydrate determinants or structurally similar determinants on the surface of immortalized bursal lymphoid stem cells participate in the adherence of these cells to the vascular regions of the bursal microenvironment. These data demonstrate that the carbohydrate structure sialyl Lewis x is developmentally regulated during chicken B cell development and may facilitate the migration of B cell progenitors to the bursal microenvironment by serving as a ligand for a lectin-like adhesion receptor.
Animals↗
Molecular basis for H blood group deficiency in Bombay (Oh) and para-Bombay individuals.
The penultimate step in the biosynthesis of the human ABO blood group oligosaccharide antigens is catalyzed by alpha-(1,2)-fucosyltransferase(s) (GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase, EC 2.4.1.69), whose expression is determined by the H and Secretor (SE) blood group loci (also known as FUT1 and FUT2, respectively). These enzymes construct Fuc alpha 1-->2Gal beta-linkages, known as H determinants, which are essential precursors to the A and B antigens. Erythrocytes from individuals with the rare Bombay and para-Bombay blood group phenotypes are deficient in H determinants, and thus A and B determinants, as a consequence of apparent homozygosity for null alleles at the H locus. We report a molecular analysis of a human alpha-(1,2)-fucosyltransferase gene, thought to correspond to the H blood group locus, in a Bombay pedigree and a para-Bombay pedigree. We find inactivating point mutations in the coding regions of both alleles of this gene in each H-deficient individual. These results define the molecular basis for H blood group antigen deficiency in Bombay and para-Bombay phenotypes, provide compelling evidence that this gene represents the human H blood group locus, and strongly support a hypothesis that the H and SE loci represent distinct alpha-(1,2)-fucosyltransferase genes. Candidate sequences for the human SE locus are identified by low-stringency Southern blot hybridization analyses, using a probe derived from the H alpha-(1,2)-fucosyltransferase gene.
ABO Blood-Group System↗