Blurring the boundaries: decays of multiparticle isomers at the proton drip line.

A multiparticle spin-trap isomer has been discovered in the proton-unbound nucleus (73)(158)Ta85 . The isomer mainly decays by γ-ray emission with a half-life of 6.1(1) µs. Analysis of the γ-ray data shows that the isomer lies 2668 keV above the known 9+ state and has a spin 10ℏ higher and negative parity. This 19- isomer also has an 8644(11) keV, 1.4(2)% α-decay branch that populates the 9+ state in (154)Lu. No proton-decay branch from the isomer was identified, despite the isomer being unbound to proton emission by 3261(14) keV. This remarkable stability against proton emission is compared with theoretical predictions, and the implications for the extent of observable nuclides are considered.

Comparison of gamma-ray coincidence and low-background gamma-ray singles spectrometry.

Aerosol samples have been studied under different background conditions using gamma-ray coincidence and low-background gamma-ray singles spectrometric techniques with High-Purity Germanium detectors. Conventional low-background gamma-ray singles counting is a competitive technique when compared to the gamma-gamma coincidence approach in elevated background conditions. However, measurement of gamma-gamma coincidences can clearly make the identification of different nuclides more reliable and efficient than using singles spectrometry alone. The optimum solution would be a low-background counting station capable of both singles and gamma-gamma coincidence spectrometry.

Tracking of airborne radionuclides from the damaged Fukushima Dai-ichi nuclear reactors by European networks.

Radioactive emissions into the atmosphere from the damaged reactors of the Fukushima Dai-ichi nuclear power plant (NPP) started on March 12th, 2011. Among the various radionuclides released, iodine-131 ((131)I) and cesium isotopes ((137)Cs and (134)Cs) were transported across the Pacific toward the North American continent and reached Europe despite dispersion and washout along the route of the contaminated air masses. In Europe, the first signs of the releases were detected 7 days later while the first peak of activity level was observed between March 28th and March 30th. Time variations over a 20-day period and spatial variations across more than 150 sampling locations in Europe made it possible to characterize the contaminated air masses. After the Chernobyl accident, only a few measurements of the gaseous (131)I fraction were conducted compared to the number of measurements for the particulate fraction. Several studies had already pointed out the importance of the gaseous (131)I and the large underestimation of the total (131)I airborne activity level, and subsequent calculations of inhalation dose, if neglected. The measurements made across Europe following the releases from the Fukushima NPP reactors have provided a significant amount of new data on the ratio of the gaseous (131)I fraction to total (131)I, both on a spatial scale and its temporal variation. It can be pointed out that during the Fukushima event, the (134)Cs to (137)Cs ratio proved to be different from that observed after the Chernobyl accident. The data set provided in this paper is the most comprehensive survey of the main relevant airborne radionuclides from the Fukushima reactors, measured across Europe. A rough estimate of the total (131)I inventory that has passed over Europe during this period was <1% of the released amount. According to the measurements, airborne activity levels remain of no concern for public health in Europe.

Gamma-ray spectroscopy at the limits: first observation of rotational bands in 255Lr.

The rotational band structure of 255Lr has been investigated using advanced in-beam gamma-ray spectroscopic techniques. To date, 255Lr is the heaviest nucleus to be studied in this manner. One rotational band has been unambiguously observed and strong evidence for a second rotational structure was found. The structures are tentatively assigned to be based on the 1/2-[521] and 7/2-[514] Nilsson states, consistent with assignments from recently obtained alpha decay data. The experimental rotational band dynamic moment of inertia is used to test self-consistent mean-field calculations using the Skyrme SLy4 interaction and a density-dependent pairing force.

Observation of a rotational band in the odd-Z transfermium nucleus 101251Md.

A rotational band has been unambiguously observed in an odd-proton transfermium nucleus for the first time. An in-beam gamma-ray spectroscopic study of 101/251Md has been performed using the gamma-ray array JUROGAM combined with the gas-filled separator RITU and the focal plane device GREAT. The experimental results, compared to Hartree-Fock-Bogolyubov calculations, lead to the interpretation that the rotational band is built on the [521]1/2(-) Nilsson state.

Collectivity and configuration mixing in 186,188Pb and 194Po.

Lifetimes of prolate intruder states in 186Pb and oblate intruder states in 194Po have been determined by employing, for the first time, the recoil-decay tagging technique in recoil distance Doppler-shift lifetime measurements. In addition, lifetime measurements of prolate states in 188Pb up to the 8+ state were carried out using the recoil-gating method. The B(E2) values have been deduced from which deformation parameters |beta2|=0.29(5) and |beta2|=0.17(3) for the prolate and the oblate bands, respectively, have been extracted. The results also shed new light on the mixing between different shapes.

Selectin ligands promote ultrasound contrast agent adhesion under shear flow.

Contrast-enhanced ultrasound imaging has shown promise in the field of molecular imaging. This technique relies upon the adhesion of ultrasound contrast agent (UCA) to targeted molecular markers of disease. This is accomplished by coating the surface of the contrast agent with a ligand that specifically binds to the intended molecular marker. Most UCA particles remain in the blood space, and their retention is influenced by the forces imposed by blood flow. For a UCA bound to a molecular target on the vascular endothelium, blood flow imposes a dislodging force that counteracts retention. Additionally, contrast agent adhesion to the molecular marker requires rapid binding kinetics, especially in rapid blood flow. The ability of a ligand:target bond complex to mediate fast adhesion and withstand dislodging force is necessary for efficient ultrasound-based molecular imaging. In the current study, we describe a flow-based adhesion assay which, combined with a novel automated tracking algorithm, enables quick determination of the ability of a targeting ligand to mediate effective contrast agent adhesion. This system was used to explore the adhesion of UCA targeted to the proinflammatory endothelial protein P-selectin via four targeting ligands, which revealed several interesting adhesive behaviors. Contrast agents targeted with glycoconjugate ligands modeled on P-selectin glycoprotein ligand 1 exhibited primarily unstable or transient adhesion, while UCA targeted with an anti-P-selectin monoclonal antibody exhibited primarily firm adhesion, although the efficiency with which these agents were recruited to the target surface was relatively low.

Nuclear isomers in superheavy elements as stepping stones towards the island of stability.

A long-standing prediction of nuclear models is the emergence of a region of long-lived, or even stable, superheavy elements beyond the actinides. These nuclei owe their enhanced stability to closed shells in the structure of both protons and neutrons. However, theoretical approaches to date do not yield consistent predictions of the precise limits of the 'island of stability'; experimental studies are therefore crucial. The bulk of experimental effort so far has been focused on the direct creation of superheavy elements in heavy ion fusion reactions, leading to the production of elements up to proton number Z = 118 (refs 4, 5). Recently, it has become possible to make detailed spectroscopic studies of nuclei beyond fermium (Z = 100), with the aim of understanding the underlying single-particle structure of superheavy elements. Here we report such a study of the nobelium isotope 254No, with 102 protons and 152 neutrons--the heaviest nucleus studied in this manner to date. We find three excited structures, two of which are isomeric (metastable). One of these structures is firmly assigned to a two-proton excitation. These states are highly significant as their location is sensitive to single-particle levels above the gap in shell energies predicted at Z = 114, and thus provide a microscopic benchmark for nuclear models of the superheavy elements.

Conversion electron cascades in 254(102)No.

The spectrum of prompt conversion electrons emitted by excited 254No nuclei has been measured, revealing discrete lines arising from transitions within the ground state band. A striking feature is a broad distribution that peaks near 100 keV and comprises high multiplicity electron cascades, probably originating from M1 transitions within rotational bands built on high K states.

Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin on leukocytes that binds to selectins. P-selectin binds to an N-terminal region of PSGL-1 that requires sulfation of at least one of three clustered tyrosines (TyrSO(3)) and an adjacent core-2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). We synthesized glycosulfopeptides (GSPs) modeled after this region of PSGL-1 to explore the roles of individual TyrSO(3) residues, the placement of C2-O-sLe(x) relative to TyrSO(3), the relative contributions of fucose and sialic acid on C2-O-sLe(x), and the function of the peptide sequence for binding to P-selectin. Binding of GSPs to P-selectin was measured by affinity chromatography and equilibrium gel filtration. 2-GSP-6, which has C2-O-sLe(x) at Thr-57 and TyrSO(3) at residues 46, 48, and 51, bound to P-selectin with high affinity (K(d) approximately 650 nm), whereas an isomeric trisulfated GSP containing C2-O-sLe(x) at Thr-44 bound much less well. Non-sulfated glycopeptide (2-GP-6) containing C2-O-sLe(x) at Thr-57 bound to P-selectin with approximately 40-fold lower affinity (K(d) approximately 25 microm). Proteolysis of 2-GP-6 abolished detectable binding of the residual C2-O-sLe(x)-Thr to P-selectin, demonstrating that the peptide backbone contributes to binding. Monosulfated and disulfated GSPs bound significantly better than non-sulfated 2-GP-6, but sulfation of Tyr-48 enhanced affinity (K(d) approximately 6 microm) more than sulfation of Tyr-46 or Tyr-51. 2-GSP-6 lacking sialic acid bound to P-selectin at approximately 10% that of the level of the parent 2-GSP-6, whereas 2-GSP-6 lacking fucose did not detectably bind; thus, fucose contributes more than sialic acid to binding. Reducing NaCl from 150 to 50 mm markedly enhanced binding of 2-GSP-6 to P-selectin (K(d) approximately 75 nm), demonstrating the charge dependence of the interaction. These results reveal a stereospecific interaction of P-selectin with PSGL-1 that includes distinct contributions of each of the three TyrSO(3) residues, adjacent peptide determinants, and fucose/sialic acid on an optimally positioned core-2 O-glycan.

Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes.

Infections of animals with parasitic worms, such as Schistosoma mansoni, induce humoral immune responses to carbohydrate antigens, raising the possibility that such antigens might be useful targets for the development of vaccines and new diagnostic approaches. Here we describe the identification of fucosylated LacdiNAc (LDNF) [GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc-R] as a new carbohydrate antigen in S. mansoni that induces humoral immune responses in infected mice. The presence of antibodies was determined by ELISA using a neoglycoconjugate synthesized to express LDNF sequences. Sera from S. mansoni-infected, but not uninfected, mice contain IgM, IgG, IgA, and IgE antibodies to LDNF. The IgG antibodies are primarily of the IgG1 and IgG3 subclasses, with no detectable levels of the complement-fixing IgG2a and IgG2b isotypes. An IgM monoclonal antibody, designated SMLDNF1, was generated from the spleens of S. mansoni-infected mice, and the antibody exhibits specific recognition of LDNF sequences, but not other fucosylated glycans tested. Immunocytochemical analysis demonstrates that LDNF antigens are localized on the tegumental surface of adult S. mansoni. Western blot analysis indicates that LDNF sequences are expressed on numerous high-molecular-weight glycoproteins from the three major human schistosome species, as well as the bird schistosome Trichobilharzia ocellata. The identification of LDNF antigen on the tegumental glycoproteins of schistosomes and the ability to synthesize LDNF conjugates should aid in the development of glycan-based vaccines and immunodiagnostic tests for schistosomiasis and in determining the role(s) of the glycans in worm development and pathogenesis.

Mice infected with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAc beta 1-->4GlcNAc) determinants.

Schistosoma mansoni is a parasitic trematode infecting humans and animals. We reported previously that adult S. mansoni synthesizes complex type biantennary N-glycans bearing the terminal sequence GalNAc beta 1-->4GlcNAc-R (lacdiNAc or LDN). We now report that mice infected with S. mansoni generate antibodies to LDN, as assessed by ELISA using a synthetic neoglycoconjugate containing LDN sequences. Sera of infected mice, but not uninfected mice, contained primarily IgM and low levels of IgG toward LDN. Interestingly, these antibodies also recognize bovine milk glycoproteins, which are known to express LDN sequences. The anti-LDN in sera of infected mice were affinity purified on immobilized bovine milk glycoproteins and shown to specifically bind LDN. An IgM monoclonal antibody (SMLDN1.1) was derived from the spleens of S. mansoni infected mice and shown to specifically bind LDN determinants. Immunoblots with affinity purified anti-LDN and SMLDN1.1 demonstrate that LDN sequences occur primarily on N-glycans of numerous glycoproteins of adult S. mansoni. LDN sequences are also expressed in many glycoproteins from S. japonicum and S. haematobium. The availability of antibody to LDN determinants should aid in defining the roles of these glycans in helminth and vertebrate biology.

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.

Efforts of health promotion teams to improve the psychosocial work environment.

This study evaluated the actions of 12 departmental "health promotion teams" and the means they used to improve the psychosocial work environment of a metal factory in a 3-year project. The teams included 80 members and were supported by organizational psychologists twice a year. A survey feedback process was applied in the beginning among all 773 employees, including management (response rate 94%). At the end, a questionnaire on perceived changes was filled out (response rate 94%). The teams guided the developmental process and initiated various activities in cooperation with the personnel. The planned activities were mainly directed at improving physical fitness and social climate. The majority of the personnel participated in these activities and were satisfied with them.

Purification and characterization of UDP-GlcNAc:Galbeta1-4GlcNAcbeta1-3*Galbeta1-4Glc(NAc)-R(GlcNAc to *Gal) beta1,6N-acetylglucosaminyltransferase from hog small intestine.

A beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni2+-chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 Glc-PA. The apparent molecular weight of the purified enzyme was 76,000 under nonreducing conditions. The enzyme has a pH optimum at 7.0 and has no requirement for any divalent metal ions. The Km values for pyridylaminated lacto-N-neotetraose and UDP-GlcNAc were 0.96 and 2. 59 mM, respectively. For its activity, this enzyme was shown to have an absolute requirement of at least a complete LacNAc (LacNAc = Galbeta1-4GlcNAc) residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal LacNAc units. The data strongly suggest that this enzyme could be involved in generating branches to central positions of preformed as well as growing polylactosamine chains, but not in synthesizing the distal branches to growing polylactosamine chains.

Applicability of survey feedback for an occupational health method in stress management.

The main stressors in work organizations have been determined in the research on mental stress. This has prompted occupational health personnel to actively look for new tools in reducing stress. However, only a few workplaces have implemented action models for health promotion by reducing stressors. The aim of this project was to investigate the applicability of survey feedback for an occupational health method of stress management. The survey feedback process, which has been one of the main approaches in organization development, was applied for stressor reduction. The employee's commitment to the programme was confirmed by participation. The occupational health personnel were responsible for carrying out the programme. The project was carried out in selected departments of one factory of an international paper company. On the basis of the survey feedback, the departments made changes in their action models, environment and instruction and guidance systems. According to the follow-up in one department, the variability of work increased, and overall mental and physical strenousness decreased. The OH personnel shifted their working model towards more active co-operation with the work units. Today the survey feedback is a routine method of the occupational health service of the company.

Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones.

Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc from the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3(+ ++GlcNAcbeta1-6)Galbeta1 -4GlcNAc and Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3Galbeta1 -4GlcNAc from the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.

Enzymatic midchain branching of polylactosamine backbones is restricted in a site-specific manner in alpha 1,3-fucosylated chains.

Branched polylactosamines on animal cell surfaces are believed to contribute to multivalent interactions in cell adhesion and cell signalling. Their biosynthesis proceeds via linear precursors that become branched by beta1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous work has identified the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (1) and the hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc (4) as acceptors for a rat serum enzyme activity (cIGnT6), which transfers GlcNAcbeta1-6 units to the midchain galactose residues. Thereby, 1 is converted to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc and 4 to the doubly branched octasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3(GlcNAcb eta1-6)Galbeta1-4GlcNAc [Leppänen, A., Salminen, H., Zhu, Y., Maaheimo, H., Helin, J., Costello, C. E., & Renkonen, O. (1997) Biochemistry 36, 7026-7036]. Here we report that neither the alpha1, 3-fucose-containing derivatives of 1 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)G lcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Gl cNAc] nor a similar derivative of 4 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)+ ++GlcNAcbeta1-3Galbeta1- 4GlcNAc] were acceptors for the rat serum cIGnT6 activity. Hence, the enzyme's branch-forming action was completely prevented at sites in the immediate neighborhood of the fucosylated loci of the polylactosamines. In Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, the inhibition of the branch-forming reaction was restricted to the fucose-carrying LacNAc unit; at the middle LacNAc, the branching proceeded normally. However, in the isomeric Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, the fucose residue prevented branching completely at the middle LacNAc and almost completely at the reducing end LacNAc. In summary, alpha1,3-fucose residues in polylactosamine chains inhibited the cIGnT6 reaction in a site-specific manner, at the fucosylated LacNAc unit itself and also at sites one and two LacNAc units upstream, but not at the LacNAc units downstream from the fucosylated locus. These data imply that site-directed branching in polylactosamines is possible in vitro with the aid of specifically positioned alpha1,3-fucosyl units, that can be removed afterward without harming the branched backbones.

The beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone.

We find that the beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) in a site-specific way to the branch-bearing hexasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (2). The product is a positional isomer of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (3), reportedly formed from 1 by another polylactosamine beta 1,6-GlcNAc transferase activity present in human serum (Leppänen et al., Biochemistry, 30 (1991) 9287). Combined use of the two kinds of activities gave in the present experiments the heptasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), in which one of the branches occupies the position of the branch in 2 and the other the position of the branch in 3.

In vitro biosynthesis of a decasaccharide prototype of multiply branched polylactosaminoglycan backbones.

Multiply branched polylactosaminoglycans are expressed in glycoproteins and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their ability to carry multiple lectin-binding determinants, which makes them prominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (5), where LacNAc is the disaccharide Gal beta1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc (1) with UDP-GlcNAc and alpha midchain beta1,6-GlcNAc transferase activity (GlcNAc to Gal), present in rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched octasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAc beta1-3'LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (2) and LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'LacNAc (3), were formed first at comparable rates. Later, both intermediates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest that, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galactoses. The enzyme-binding epitope of 1 is probably LacNAc beta1-3'LacNAc, because the trisaccharides GlcNAc beta1-3'LacNAc and LacNAc beta1-3Gal as well as the tetrasaccharide GlcNAc beta1-3'LacNAc beta1-3Gal were poor acceptors, while LacNAc beta1-3'LacNAc was a good one. Midchain beta1,6-GlcNAc transferase activities present in serum of several mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. We suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.