Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

A monoclonal antibody detecting an Ia specificity mapping in the I-A or I-E subregion

An H-2^s anti-H-2^d monoclonal la antibody (CE197) is described which appears to detect a new Ia specificity that maps either in the I-A or the I-E subregion, depending on the haplotype examined. Binding inhibition studies with radiolabeled, monoclonal I-A-and I-E-specific antibodies imply that CE197 reacts with a public determinant shared by a variety of A -A and E -E dimers.     

A new α2HS-glycoprotein typing by isoelectric focusing

Summary Isoelectric focusing (pH 4.0–5.0) of serum ₂HS-glycoprotein on polyacrylamide gels has been found to be a useful tool in population genetics and forensic science. Using this method, we isolated three common types, ₂HS 1-1, ₂HS 2-1 and ₂HS 2-2, and showed that ₂HS types are determined by two autosomal codominant alleles, ₂ HS ¹ and ₂ HS ². The method is simple, fast and easy to perform. Results of typing for the two alleles, ₂ HS ¹ and ₂ HS ², are described for a Japanese population sample (n=1003).     

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic ( and ) and regulatory () subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of free CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with and subunits of Xenopus CK2, with the subunit of D. rerio, and with Xl CK2A¹⁵⁶, which although inactive can bind tightly to CK2, and with Xl CK2E⁷⁵E⁷⁶, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2 or CK2E⁷⁵E⁷⁶ in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2, the activity increased further to 15–20-fold of the controls. Transfection of CK2 alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2A¹⁵⁶ yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 or CK2E⁷⁵E⁷⁶ with CK2A¹⁵⁶ caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 subunits. These results can be interpreted as meaning that CK2A¹⁵⁶ is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 subunit. Addition of recombinant CK2 to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 subunit is limiting in the extracts and that an excess of free CK2 has been produced in the transfected cells. Transfection of cells with CK2E⁷⁵E⁷⁶ results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2E⁷⁵E⁷⁶ to heparin increases considerably when it forms part of the holoenzyme CK2₂₂.     

The expression of I-Ed molecules in F1 hybrid mice detected with antigen-specific,I-Ed-restricted cloned T-cell lines

The expression of polymorphic determinants on I-E molecules is largely dependent on allelic variation in the E chain. We have previously analyzed the expression of E ^k and E ^b chains in F₁ hybrid mice by a combination of techniques, and have shown that functional variation detected by the responsiveness of cloned T-cell lines specific for these molecules correlates well with serological determination of E expression. In the present study, we have extended our analysis to E ^d expression in F₁ hybrid mice. We show that E ^d is relatively poorly expressed in three F₁ combinations: H-2 ^d× H-2 ^b, H-2 ^d× H-2 ^s, and H-2 ^d× H-2 ^u. The former two crosses express E chains from the H-2 ^dparent only; when recombinant strains carrying E ^b or E ^s and an active E gene are used, E ^d expression is significantly increased. On the other hand, H-2 ^umice synthesize E chains; the poor expression of E ^d chains in this F₁ hybrid apparently reflects the strong preferential association of E ^u chains with all E molecules thus far analyzed. These results confirm that E chains compete for binding to E chains and that preferential association of different allelic forms of E chains with E chains is a generalized phenomenon. They also illustrate the importance of the rate of biosynthesis of Ia chains for cell-surface expression.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functionalin vivo

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete ^2FS precursor protein or only for the subunit have been placed under the control of the P_R promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the P_R promoter, the complete ^2FS constructions constitutively express a smaller polypeptide of 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (²P_F and ²P_S) located in the duplicated ^2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp² cDNA sequence, when fused upstream to the cDNA coding for ¹-antitrypsin, constitutively promotesin vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against ¹-antitrypsin or haptoglobin.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Structural comparisons of serologically identical IA- and IE-encoded antigens from inbred and wild mice

The IA and IE products of B10.S(9R), B10.A, B10.KPB128, and B10.GAA37 were analyzed for primary structural variations by comparative tryptic peptide mapping. The A,A , andE products of B10.S(9R) and B10.A differed in about 40% of their acid-soluble tryptic peptides, indicating that intra-I-region recombinant strain B10.S(9R) received the genes encoding A, A, and E from theH- 2 ^s parental chromosome rather than fromH- 2 ^a . The tryptic peptides of E chains from B10.S(9R) and B10.A were indistinguishable, suggesting that B10.S(9R) received the gene encoding the E chain from theH- 2 ^a parental chromosome. Consistent with the results of others, these data suggest that the genes encodingA ,A and E chains are centromeric to theIJ subregion, while the gene encoding E chains is telomeric toIJ. The I-region products of two congenic lines carrying wild-derivedH- 2 haplotypes on a C57BL/10 background, designated B10.KPB128 and B10.GAA37, are serologically indistinguishable from those of B10.S(9R). The IA and IE products of B10.S(9R) were compared with those of B10.GAA37 and B10.KPB128 to determine the structural similarity of serologically identical products from allopatric populations of wild mice. The A,A , and E products of B10.S(9R) were indistinguishable from those of B10.GAA37 and B10.KPB128 by comparative tryptic peptide mapping. The E chains of these three lines differed in one or two of their acid-soluble tryptic peptides. The results indicate that the IA-encoded products of these three lines are structurally very similar and may be identical suggesting that some alleles of the A, A, and E chains may be maintained in stable linkage associations in allopatric populations of wild mice. The minor structural variations detected in the E chains of these three congenic lines indicate that the E chain is encoded by chromosome 17 and suggest that allelic E chains exhibit considerably less structural variability than other I-region encoded antigens.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

A note on dimensionless life histories for birds versus mammals

Summary Offspring production over the adult lifespan (b/M whereb is the yearly fledgling or offspring production and 1/M is the mean adult lifespan) is an approximate invariant within both birds and mammals. The two taxa differ, however, in that mammals have bothM and b as invariants (b/M = b/M) while birds do not ( is the age at first breeding). Birds have a surprising cancellation in that bothM andb are ^–0.25.     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

The minimal polymorphism of class II Eα chains is not due to the functional neutrality of mutations

Given the extensive allelic amino acid sequence polymorphism present in the first domain of A, A, and E chains and its profound effects on class II function, the minimal polymorphism in the mouse E chain (and in its human homologue DR) is paradox. Two possible explanations for the lack of polymorphism in E are: (1) the E chain plays such a uniquely critical structural/functional role in antigen presentation, T-cell activation, repertoire selection, and/or pairing with E or other proteins for expression that it cannot vary, and mutations are selected against; (2) the E chain plays a less significant role than the outer domains of other major histocompatibility complex (MHC) proteins in determining the interactions with processed peptides or with T-cell receptor (TCR), so there is no selective pressure to maintain new mutations. To explore this question we compared the ability of transfectants expressing wild type (wt) EE ^d and mutant E wt E ^d proteins to present peptides and bacterial superantigens to T-cell hybridomas. Mutations at the E amino acid positions 31, 52, and 65&66, to residues that represent allelic alternatives in A chains, significantly reduced activation of peptide-specific T hybridomas, and mutations at 71 sometimes enhanced T-cell stimulation. None of the E mutations reduced, and some enhanced, superantigen stimulation of T-cell hybridomas. These results argue against the hypothesis that E chains are minimally polymorphic because mutations in E are functionally neutral.     

Lectin histochemistry of complex carbohydrates in human cervix

Summary Complex carbohydrates in the human cervix were studied histochemically using lectins, conjugated to horseradish peroxidase and correlated procedures. Stratified squamous epithelium of the exocervix and columnar epithelium of the endocervix in some, but not all specimens showed staining for terminal -N-acetyl-d-galactosamine, -d-galactose, -d-galactose and -l-fucose. the staining for -N-acetylgalactosamine and -galactose, the terminal sugars in blood group A and B antigens respectively, corresponded to a large extent with ABO blood type. One exception was the lack of staining for terminal -N-acetylgalactosamine in endocervical secretions in three of nine blood type A patients. A second exception was the staining for terminal -galactose in endocervical secretions in about half of blood type O and A specimens. The type and amount of glycoprotein formed by endocervical columnar cells differed according to location in superficial compared with deep portions of the glands and according to location at the junction with exocervix compared with the more internal regions. Staining of endothelial cells for blood group A and B antigens was confined to subjects of blood type A and B respectively, although three of nine type A specimens showed no lectin reactivity for group, A antigen. Endothelial cells evidenced affinity forUlex europeus I agglutinin demonstrative of fucose in all specimens. Mast cells disclosed lectin affinity consistent with the presence of terminal or internal mannose orN-acetylglucosamine residues. Two blood type O specimens were examined with conjugated lectins at the ultrastructural level. Secretory granules stained for content of terminal -galactose, -galactose and fucose. These results support and concur with biochemical studies of complex carbohydrates in human cervical tissues. They reveal, in addition, the location of the blood group antigens in the human exocervix and endocervix and the marked heterogeneity among endocervical columnar cells in glycoprotein production.     

Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence

A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14 (A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104 (G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer,pH 7.0, at 5°C, and the structural flexibility of-apohemoglobin, in the absence and presence of human-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (E ^O) and Förster distances (R ₀ ^O ) were analyzed by least-squares to determine the goodness of fit ( _R ² ) for the assumed distribution parameters: average distance ¯r and half-widthhw. Data for-apohemoglobin in the absence and presence of ^h chains yielded values for ¯r of 18 and 22 Å andhw of 20 and 8.5 Å, respectively. Although the increase in ¯r for-apohemoglobin in the presence of ^h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.     

DNA sequence analysis of a rat RT1 class 11 A β gene

The rat major histocompatibility complex (RT1) encodes twin sets of class II molecules, each consisting of two polypeptide chains referred to as A and A , and E and E . A gene encoding the RTLA chain was isolated from a rat genomic library using an HLA-DQ chain cDNA as a probe. The nucleotide sequence of the coding regions of this gene was determined. Comparison of this sequence with those of the corresponding genes of mouse (H-2A ) and human (HLA-DQ ) revealed that this gene has been highly conserved during evolution, and that some parts of the molecule are more conserved than others. Analysis of the nucleotide sequence encoding the two external domains suggests that the membrane proximal domain has been subject to conservative selection, whereas replacement substitutions have been selected positively at certain residues within the amino terminal domain. The overall organization of the RT1.A gene is similar to that of the H-2A gene.     

Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with 1,3-fucosyltransferase activity and had a specific activity of 5–6 µmol min^–1 mg^–1 and anM _r 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal1-4GlcNAc, NeuAc2-3Gal1-4GlcNAc and Fuc1-2Gal1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc1-2Gal1-4Glc and the Type 1 compound Gal1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc2-6Gal1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major 1,3-fucosyltransferase gene expressed in human liver.Died June, 1991     

Constant-time NOESY: An aid in the analysis of protein NMR spectra

Summary A constant-time version of the homonuclear NOESY experiment (CT-NOESY) is described. The experiment yields simplified protein spectra, in which cross peaks arising from protons with zero or small couplings are differentiated from other cross peaks, thus partially overcoming the problem of signal overlap. In addition, the CT-NOESY spectrum provides information on the magnitude of³J_NH- and³J coupling' constants, and is thus useful to determine torsion angle constraints and to perform stereospecific assignments of CHH protons in the case of³J constants.