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The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette-Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-gamma, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.

Background: Acute otitis media is one of the most commonly-diagnosed childhood infections. This study assessed the efficacy of a novel vaccine that contained polysaccharides from 11 different Streptococcus pneumoniae serotypes each conjugated to Haemophilus influenzae-derived protein D in prevention of acute otitis media., Methods: 4968 infants were randomly assigned to receive either pneumococcal protein D conjugate or hepatitis A vaccine at the ages of 3, 4, 5, and 12-15 months and were followed-up until the end of the second year of life. Middle-ear fluid was obtained for bacteriological culture and serotyping in children who presented with abnormal tympanic membrane or presence of middle-ear effusion, plus two predefined clinical symptoms. The primary endpoint was protective efficacy against the first episode of acute otitis media caused by vaccine pneumococcal serotypes. Analysis was per protocol., Findings: From 2 weeks after the third dose to 24-27 months of age, 333 clinical episodes of acute otitis media were recorded in the protein D conjugate group (n=2455) and 499 in the control group (n=2452), giving a significant (33.6% [95% CI 20.8-44.3]) reduction in the overall incidence of acute otitis media. Vaccine efficacy was shown for episodes of acute otitis media caused by pneumococcal vaccine serotypes (52.6% [35.0-65.5] for the first episode and 57.6% [41.4-69.3] for any episode). Efficacy was also shown against episodes of acute otitis media caused by non-typable H influenzae (35.3% [1.8-57.4]). The vaccine reduced frequency of infection from vaccine-related cross-reactive pneumococcal serotypes by 65.5%, but did not significantly change the number of episodes caused by other non-vaccine serotypes., Interpretation: These results confirm that using the H influenzae-derived protein D as a carrier protein for pneumococcal polysaccharides not only allowed protection against pneumococcal otitis, but also against acute otitis media due to non-typable H influenzae. Whether this approach would also allow improved protection against lower respiratory tract infections warrants further investigation.

The safety, immunogenicity, and protective efficacy of two non-replicating antigen-based vaccines and one live-attenuated virus (LAV) vaccine for dengue type-2 (dengue-2) virus were evaluated in the rhesus macaque model. The non-replicating vaccines consisted of whole, purified inactivated virus (PIV) and a recombinant subunit protein containing the amino-(N)-terminal 80% of envelope protein (r80E), each formulated with one of five different adjuvants. Each formulation was administered to three animals on a 0, 3-month schedule. Following the primary immunizations, 37 of 39 animals demonstrated dengue-2 virus neutralizing antibodies. After the booster immunizations all animals had dengue neutralizing antibodies with peak titers ranging from 1:100 to 1:9700. The highest neutralizing antibody titers were observed in the groups that received r80E antigen formulated with AS04, AS05, or AS08 adjuvant, and PIV formulated with AS05 or AS08 adjuvant. These newer adjuvants are based on alum, fraction QS-21 of saponin, and monophosphoryl lipid A (MPL). Protection was evaluated by dengue-2 virus challenge 2 months after the booster by the measurement of circulating virus (viremia) and post-challenge immune responses. Several groups exhibited nearly complete protection against viremia by bioassay, although there was evidence for challenge virus replication by Taqmantrade mark and immunological assays. None of the vaccines conferred sterile immunity.

We have demonstrated that recombinant hepatitis E vaccine suitable for clinical evaluation was highly immunogenic and efficacious in preventing hepatitis E and even infection in rhesus macaques following intravenous challenge with three different genotypes of hepatitis E virus (HEV). Two doses of vaccine were essential for optimal protection; the two-dose regimen was more important than the formulation of the vaccine for achieving efficacy. The titers of anti-HEV that were protective in this study were quantified against a World Health Organization (WHO) standard. This permits direct comparison of the results with other studies. The results of this pre-clinical trial of a candidate hepatitis E vaccine strongly suggest that it will be highly efficacious for preventing hepatitis E in the field trial of this vaccine that is currently in progress in Nepal.

The mechanisms by which administration of a formalin-inactivated respiratory syncytial virus vaccine resulted in enhanced disease among children after they later became naturally infected with the virus remains largely undefined. After immunization and live virus challenge, the cotton rat demonstrated the histopathologic marker of the enhanced disease, polymorphonuclear leukocyte infiltration of lung alveolar spaces. We now report that immunization with formalin-inactivated vaccine formulated with the adjuvant, 3-deacylated monophosphoryl lipid A, dramatically reduces or eliminates the polymorphonuclear leukocyte infiltration within the alveoli of cotton rats post-challenge. We suggest, that this or similar adjuvants may be beneficial components of candidate non-replicating respiratory syncytial virus vaccines, whose development has been hampered by safety concerns.

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

Infection of the cotton rat lung with a human strain of respiratory syncytial virus results in substantial virus replication and is associated with mild-to-moderate peribronchiolitis, perivasculitis, and bronchitis. Reinfection after 49 days did not result in detectable virus replication, but surprisingly, was associated with an earlier appearance and accentuation of the three types of lesions seen in cotton rats undergoing primary infection. Animals primed with formalin-inactivated virus and challenged after 49 days had pulmonary viral titers 1/10 to 1/100 of that seen in naive animals, but developed markedly accentuated lesions of the same type as in animals undergoing primary or secondary infection. In addition, the animals with the vaccine-enhanced disease developed alveolitis and interstitial pneumonitis, which seem to be specific markers for the vaccine enhancement. These latter markers may be useful in determining the safety of nonreplicating vaccines.

The ability of monophosphoryl lipid A (MPL), QS-21 and alum to alter the immunologic response to immunization with respiratory syncytial virus a chimeric FG construct (FG) subunit vaccine was examined in BALB/c mice. FG/MPL, FG/alum, and FG/MPL/QS-21 combinations increased non-neutralizing antibody response, while FG/QS-21 did not. FG subunit vaccine with MPL, QS-21, or both had cytokine responses more closely resembling primary infection than FG/alum, with decreased interleukin-4 mRNA levels and increased IgG2a isotype antibody. The lungs of the mice immunized with FG subunit vaccines showed a heightened inflammatory response to respiratory syncytial virus challenge as compared to live virus immunization. Adjuvants can be used to alter the humoral and cellular responses to RSV subunit immunization.

Human recombinant myeloperoxidase was evaluated in a cell-free system for its inactivation properties on the replication of human immunodeficiency virus, HTLV-IIIB. In the presence of a hydrogen peroxide generating system (glucose and glucose oxidase) and sodium thiocyanate, the recombinant enzyme inhibited virus-induced syncytium formation and viral replication without causing any cytopathic effects on SupT1 reporter cells. In addition, U937 monocytoid cells, chronically infected with HIV1, were exposed to recombinant myeloperoxidase (10 U/ml) and monitored during 48 h for the accumulation of intracellular p24 viral antigen. Under these conditions, the recombinant enzyme significantly reduced intracellular viral replication without affecting cell viability.

Beta-Galactosidase from Saccharomyces lactis was found to be able to catalyze both the anomerization of alpha-lactose and the hydrolysis of beta-lactose; the rate of hydrolysis appeared to be four times higher with a 1:1 mixture of alpha and beta lactose than with a freshly prepared solution of alpha-lactose. The enzyme was also found to be unable to hydrolyze alpha-lactose. Thus, it appears that beta-galactosidase from S. lactis has its hydrolytic activity on lactose adapted only to the naturally more abundant beta-lactose.

In order to obtain a better understanding of the possible influence of the primary sequence of a protein on its folding pathway, renaturation of reduced human milk lysozyme was compared to that of reduced hen egg white lysozyme. Following disulfide bond formation, under identical conditions, similar products were found during the folding of both lysozymes, but the kinetics of appearance and disappearance of these intermediates as well as the appearance of the native conformation were different.

The Lewis blood group-specified N-acetylglucosaminide alpha 1 goes to 4 fucosyltransferase and an N-acetylglucosaminide alpha 1 goes to 3 fucosyltransferase have been copurified over 500,000-fold from human milk by affinity chromatography on GDP-hexanolamine agarose. The purified enzyme preparation migrates as two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr = 53,000 and 51,000. Analysis of the acceptor substrate specificity of the transferase(s) and structural characterization of the reaction products indicate that the enzyme(s) forms the Fuc alpha 1 goes to 4GlcNAc, Fuc alpha 1 goes to 3GlcNAc, and Fuc alpha 1 goes to 3Glc linkages with oligosaccharide acceptors containing the nonreducing terminal sequences Gal beta 1 goes to 3GlcNAc, Gal beta 1 goes to 4GlcNAc, and Gal beta 1 goes to 4Glc, respectively. The two fucosyltransferase activities are activated to the same extent by a variety of divalent metal ions, inactivated at identical rates by thermal denaturation or reaction with N-ethylmaleimide, and inhibited to the same extent by rabbit antiserum prepared against the purified fucosyltransferase(s). In addition, kinetic analysis of the initial rate data obtained using acceptors for one of the fucosyltransferase activities as an inhibitor of the second suggests that acceptors for both fucosyltransferase activities bind at a single active site.

Evidence is presented suggesting that hepatocytes contain a receptor that binds glycoproteins specifically through fucose in alpha1-->3 linkage to N-acetylglucosamine. Human lactoferrin, which contains this type of linkage, is rapidly cleared from the circulation of mice after intravenous injection, and greater than 90% of the injected material is found in hepatocytes. Binding of lactoferrin is mediated through its carbohydrate groups, since its clearance is prolonged after periodate oxidation or after its oligosaccharide groups are extensively degraded with glycosidases. In addition, glycopeptides from lactoferrin inhibit lactoferrin clearance. That lactoferrin clearance is mediated through binding to its fucosyl groups is suggested for several reasons. First, transferrin and asialotransferrin, whose oligosaccharide groups are essentially structurally identical to those of lactoferrin but devoid of fucose, are not cleared on intravenous injection. Second, when fucose is incorporated into asialotransferrin by alpha1-->3 N-acetylglucosamine fucosyl transferase, the resulting fucosylated derivative is cleared rapidly. Neither mannan nor derivatives of orosomucoid that are cleared by binding to receptors for galactose, N-acetylglucosamine, or mannose, inhibit clearance of lactoferrin although clearance is inhibited by fucoidin. Finally, glycoproteins containing fucose in alpha1 --> 2 linkage to galactose or alpha1 --> 6 linkage to N-acetylglucosamine do not inhibit lactoferrin clerance by the liver. Since clearance of other glycoproteins, such as human lactoperoxidase, also appears to be mediated through binding to the same hepatocyte receptor as lactoferrin, it is concluded that the fucose-specific receptor studied here may fulfill other functions than binding lactoferrin. Preliminary studies with liver homogenates and detergent extracts of liver show binding in vitro.

Lactoferrin (LF) has been recently proposed as a physiologic regulator of the granulocyte monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit CFU-GM growth by decreasing production and release of colony stimulating activity (CSA) by monocytes and macrophages. Human milk LF saturated with iron, at concentrations ranging from 10(-18) to 10(-8) M was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of LF within the culture system used, no significant inhibition of CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4-day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of LF was the same. Various possible explanations for not confirming the reported inhibiting activity of iron saturated LF were explored: 1) masking inhibition of the system by prostaglandin E2 (PGE2), 2) masking inhibition of the system by bovine LF still detectable in the fetal calf serum after heating, 3) preinhibition of the system by leukemic-associated inhibitory activity (LIA) possibly present in the culture system, 4) the iron and calcium content of the culture medium used, 5) the fixation of LF to plastic compounds, 6) the source of the human LF used, 7) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of LF and thus no evidence was found for a significant role of LF in the regulation of CSA production by monocytes. Peripheral blood human monocytes isolated by elutriation and incubated in albumin free medium in the presence of either 125I-LF or colloidal gold-labeled LF showed no LF binding.

Human embryonic skin fibroblasts attach and spread on surfaces on which a fucosyltransferase purified from human milk has been immobilized. The adhesion-enhancing effect of the transferase involves specific interactions of the enzyme surface with the cell surface carbohydrate acceptors, as suggested by the following findings. About 80% of human embryonic skin fibroblasts attach and spread in 1 hr on fucosyltransferase surfaces; in contrast, bovine serum albumin, fetuin, asialofetuin, and asialotransferrin surfaces fail to enhance adhesion. The adhesion-mediating activity of the transferase is destroyed by alkylation of the sulfhydryl groups or by heating. The adhesion on fucosyltransferase surfaces is inhibited by glycoprotein, glycolipid, and oligosaccharide acceptors containing the sugar sequence galactosyl-(beta 1 leads to 4)-N-acetylglucosamine, in agreement with the substrate specificity of the enzyme. The results suggest that glycosyltransferases are able to stimulate cell adhesion in a manner similar to that proposed for lectins.

Using galactosylated bovine serum albumin coupled to colloidal gold (galBSA-CG) as a probe, the receptor-mediated endocytosis pathway has been analyzed qualitatively and quantitatively at the ultrastructural level in cultured rat hepatocytes. The results showed that galBSA-CG was specifically recognized by the asialoglycoprotein receptor of the hepatocytes, thus confirming biochemical findings. The probe was preferentially bound in coated pits of the cell surface. When bound elsewhere on the plasma membrane, it apparently moved towards coated regions. In both cases, it was then internalized via coated vesicles. The galBSA-CG passed through pleiomorphic tubular structures and in endosomes, a pool of smooth-surfaced vesicles of various size (50-350 nm), before transiently accumulating in multivesicular bodies. The latter then fused with lysosomes where the glycoproteinic moiety of the probe was degraded, as judged by the flocculated aspect of the accumulated gold particles. About 10% of the internalized ligand was recycled back to the cell surface via secreting vesicles containing lipoprotein-like particles without having apparently passed through lysosomes, which suggests the existence of a pre-lysosomal sorting mechanism of the endocytosed material. Functional recovery of the morphologically restored biliary polarity of hepatocytes in culture was indicated by the fact that galBSA-CG finally appeared in the reconstituted bile canaliculi.

In vitro, renaturation of reduced and unfolded lysozyme is catalyzed by a mixture of reduced and oxidized glutathione. After initiation of disulfide bond formation associated with the folding process of reduced human lysozyme, molecules have been trapped in a stable form with iodoacetic acid (preserving disulfide bonds) at various times of reoxidation. Each population of molecules trapped in this way was then analyzed by acrylamide gel electrophoresis which separates intermediates on the basis of the number of disulfide bonds they contain and the mean volume of the polypeptide chain. Moreover, the rate of reoxidation of the regeneration mixture was monitored by changes in enzymatic activity, fluorescence quantum yield, and global sulfhydryl group titer. Enzymatic activity was observed to appear after an induction period, and no intermediate, except the fully regenerated species, is active. The first two disulfide bonds reoxidize rapidly, and very few intermediates containing one or two disulfide bonds could be trapped. On the other hand, the intermediates containing three and four disulfide bonds are more predominant, and their formation proceeds more slowly. A folding pathway is suggested, based on the kinetic studies of appearance and disappearance of the various observed intermediates. When these results are compared with those obtained for hen egg white lysozyme and with those found in literature, it can be concluded that the reduced human protein recovers its native conformation more progressively and with more difficulty than the hen egg white protein. This difference might be explained by a greater organization and a greater hydrophobicity in the human molecule.

When the cell lysis of Micrococcus luteus by hen egg white or human lysozyme is performed in the presence of bovine or human lactoferrin, a temporary increase of the turbidity of the solution as followed at 450 nm is observed. Examination of the suspension under light microscopy has proven that the protoplasts produced upon lysozyme action are agglutinated by lactoferrin. The rate of agglutination depends on pH, lactoferrin, lysozyme and cells concentrations. Agglutination is maximal at pH 5.5. Around 1.4 X 10(6) binding sites for lactoferrin per cell have been determined through a Scatchard plot analysis. The binding to the cells is not mediated by the glycosidic moiety of lactoferrin but rather by a charge-to charge interaction as succinylation of about four out of the 39 lysines of lactoferrin completely abolishes its ability to agglutinate the cells. Binding does not depend on ionic iron nor on the iron content of lactoferrin itself.

In the search for the biochemical basis of the control of glycosylation of cell surface carbohydrates, revertant clones were isolated from previously characterized wheat germ agglutinin-resistant clones of B16 mouse melanoma cells by selection for resistance to Lotus tetragonolobus lectin or to ricin. Comparison of the wheat germ agglutinin-resistant clones with the parent and revertant clones indicated that this phenotype was correlated with an increased sensitivity to the Lotus lectin, a 60- to 70-fold increase in alpha 1 leads to 3 fucosyltransferase activity and a decreased sialic acid content of the N-glycosidic chains of glycoproteins. The results suggest a novel type of control mechanism for lectin resistance, an increase in a glycosyltransferase activity. The presence of alpha 1 leads to 3 bound fucose on N-acetylglucosamine residues would interfere with the addition of sialic acid by alpha 2 leads to 3 linkages to galactose residues in the carbohydrate units, and this change could explain the resistance to wheat germ agglutinin and the increased sensitivity to the Lotus lectin. A change in a regulatory gene for the fucosyltransferase as a possible primary cause for the changed phenotype is discussed.

beta(1-4)-Galactosyltransferase from human milk (the A protein of lactose synthase) has been found to be heterogeneous when fractionated by affinity chromatography against insolubilized alpha-lactalbumin, using a linear gradient of decreasing N-acetylglucosamine concentration. Three forms were isolated. Molecular weights of the different species, as determined by sodium dodecylsulphate gel electrophoresis, were found to be 38 000, 43 000 and 50 000. The 38 000 and 50 000 species were studied for their catalytic ability to synthesize either lactose in the presence of alpha-lactalbumin, or N-acetyllactosamine in the presence and absence of the 'specifier' protein. Appreciable difference was observed between the two enzyme forms with respect to their catalysis of lactose synthesis with alpha-lactalbumins from various sources. Differences in the rate of production of N-acetyllactosamine in the presence of alpha-lactalbumin were also observed. For the lowest-molecular-weight species it was found that the inhibitory effect of alpha-lactalbumin upon N-acetyllactosamine synthesis becomes an activating effect at higher alpha-lactalbumin concentrations, while no such inversion was observed for the other species. The results suggest that the conformation at the site of association of the enzyme with the acceptor saccharide or alpha-lactalbumin has been changed, presumably by a pratial enzymic hydrolysis.

The hepatic uptake and degradation of human diferric 125I-lactoferrin by the liver of the intact rat were studied. After intravenous injection, the tracer was rapidly cleared by the liver, probably by adsorptive pinocytosis, as inferred from observations with a 3,470-fold dose range. Endocytosed lactoferrin was transferred, with a delay, from a light-density subcellular particle to an organelle that had a density similar to lysosomes. The loss of protein bound 125I from the liver was very slow (half-life 2.7 h), and its rate matched closely that of human asialotransferrin type 3. Lactoferrin was found to be a poor substrate for lysosomal hydrolases in vitro. Fucoidin effected the release of a portion of lactoferrin from the liver back into the plasma. By using this agent, indirect evidence was obtained suggesting that a fraction of lactoferrin is being repeatedly endo- and exocytosed (diacytosed) by the liver over prolonged periods of time. Fucosylation failed to impart lactoferrinlike properties on human asialotransferrin type 1, although the derivatized protein exhibited a less than or equal to 10-fold increase in affinity for the liver relative to the parent molecule.

Four rabbit antibodies have been prepared, which are specifically directed against alpha-lactalbumins from different sources; namely human, cow, goat and sheep milk. Each of these antibodies was tested for its ability to react with, separately, each of the four proteins. The immunological reactions were assessed by means of different techniques: double immunodiffusion in agar gel as well as affinity chromatography of antibodies, using antigens covalently bound to an insoluble matrix. In each case, the strongest reaction was observed between homologous antibody and (matrix-bound) antigen; heterologous antigens were, however, also capable of cross-reaction. Whereas no cross-reaction between human alpha-lactalbumin and antibodies against the bovine protein could be evidenced by immunodiffusion, the occurrence of soluble complexes has been disclosed by means of a gel filtration technique.

In an effort to assess the effect of surface carbohydrates upon the metastasizing properties of tumor cells, lectin-resistant mouse melanoma cells were selected. Wheat-germ-agglutinin-resistant lines displayed mainly decreased metastasis properties as well as well-defined alterations in surface carbohydrates: in a glycopeptide with four side chains, two of them were missing their terminal sialic acid residues while two fucoses were newly attached to the oligosaccharide. The enzymatic defect could be pinpointed to an over-60-fold increase in fucosyltransferase, while the sialyltransferase did not decrease significantly. Revertants were again selected with lectins and their fucosyltransferase activities returned to normal values again. The metastasizing potential of the revertants was not yet assessed carefully but a return of some of the metastasizing potential was noted.

Intrinsic as well as extrinsic fluorescence of an immobilized protein was used for the study of the interactions between alpha-lactalbumin-Sepharose and protein ligands. The fluorescence peak of the human alpha-lactalbumin-agarose conjugate was shifted to the blue and quenched in the presence of the galactosyl transferase (A-protein), indicating the probable formation of a complex between both proteins. The natural fluorescence of human alpha-lactalbumin bound to Sepharose was specifically quenched in presence of antihuman alpha-lactalbumin antibodies. This change in fluorescence appears to be due to binding of the antibodies to the immobilized antigen. Furthermore, the extrinsic fluorescence of a bound dye such as 2-p-toluidinylnaphthalene-6-sulfonate was used to confirm the existence of binding between antibodies and alpha-lactalbumin-agarose, and to obtain values for the association constant. A value of 5.6-10(+6) M(-1) for the binding constant was reported, which compares favorably with other data obtained by equilibrium dialysis.

1. Various insoluble alpha-lactalbumins (bovine, bovine glyco-alpha-lactalbumin, human and human nitrated) have been prepared by coupling these proteins on to an agarose gel with use of cyanogen bromide. 2. Some intrinsic fluorescence properties, such as fluorescence maximum and pH dependence, were considered in order to study conformational changes of the alpha-lactalbumins covalently bound to an insoluble matrix. Examination of the pH-fluorescence profiles as well as the position of the maximum in the emission spectrum indicates that the Sepharose matrix does not appreciably modify the conformation of human and bovine glyco-alpha-lactalbumins. Some changes in the fluorescence spectrum (peak shifting towards longer wavelength) was observed for bovine alpha-lactalbumin and appeared to be due to alteration of the environment of the tryptophan side-chains in the protein upon coupling to the agarose gel. The emission spectrum of the insolubilized human nitrated alpha-lactalbumin indicates that the polypeptide chain of this protein gained some native conformation when covalently bound to the carrier. 3. The extrinsic fluorescence of a bound dye, such as 2-p-toluidinylnaphthalene-6-sulfonate, was used to study and to compare the hydrophobic sites on the surface of insoluble alpha-lactalbumins with the same proteins in solution. Considering the fluorescence properties of the protein with dye complexes it was found that both states of alpha-lactalbumins (insoluble and free in solution) bind the dye with similar association constants. However, the positions of the maxima in the emission spectra are all somewhat shifted towards longer wavelengths, suggesting that the dye binding site is located in a more polar environment when the proteins are bound to agarose. The human nitrated alpha-lactalbumin retains about equal possibility of binding this fluorescent dye. 4. As shown for bovine alpha-lactalbumin in solution, the binding of 2-p-toluidinylnaphthalene-6-sulfonate towards the various insoluble alpha-lactalbumins was not appreciably modified by the presence of various small compounds such as sugars and UDP, which are effectors in the lactose synthetase function.

Through the use of affinity chromatography, a homogeneous preparation of human beta(1 leads to 4)-D-galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human alpha-lactalbumin. A kinetic analysis of the transfer of D-galactose to N-acetyl-D-glucosamine and the beta(1 leads to 4) linked N-acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor moleucles. Furthermore, experiments with Na-acetylglucosamine-beta(1 leads to4)-N-acetylmuramic-pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent Km values for A protein) as galactose acceptors. In contrast to its ability to inhibit N-acetyllactosamine production, alpha-lactalbumin did not inhibit the transfer of D-galactose to the N-acetylglucosamine oligomers or the glycopeptides. Although alpha-lactalbumin can switch the specificity of A protein from N-acetyl-D-glucosamine to D-glucose resulting in the production of lactose, no transfer of galactose was observed to beta(1 leads to 4)-linked glycose oligomers or to a collagen glycopeptide, D-glycopyranosyl-alpha(1 leads to 2)-D-galactopyranosyloxy-beta(1 leads to 5)-lysine. IT therefore appears that alpha-lactalbumin can only modify human A protein for monosaccharide acceptors.

Lactoferrin has been proposed recently as a physiological regulator of the granulocyte-monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit the CFU-GM growth by decreasing production and release of colony stimulating activity by monocytes and macrophages. Human milk lactoferrin saturated with iron, at concentrations ranging from 10(-8) M, was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of lactoferrin within the culture system used, no significant inhibition of the CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4 day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of lactoferrin was the same. Various possible explanations for not confirming the reported inhibiting activity of iron-saturated lactoferrin were explored: (a) masking inhibition of the system by prostaglandin E2 (PGE2), (b) masking inhibition of the system by bovine lactoferrin present in the fetal calf serum, (c) preinhibition of the system by leukemic-associated inhibitory activity possibly present in the culture system, (d) the iron and calcium content of the culture medium used, (e) the fixation of lactoferrin to plastic compounds, (f) the source of the human lactoferrin used, and (g) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of lactoferrin and thus no evidence was found for a significant role of lactoferrin in the regulation of human granulopoiesis.

A fucosyltransferase was solubilized by extraction with Triton CF-54 from a wheat-germ agglutinin-resistant variant of mouse B16 melanoma. Through affinity chromatography on GDP hexanolamine--Sepharose a 44-fold enrichment of its specific activity was obtained. Analysis of its specificity indicated that the enzyme is an N-acetylglucosaminide 3-alpha-L-fucosyltransferase, which is able to transfer fucose to oligosaccharides containing Gal(beta 1-4)GlcNAc and Gal(beta 1-4)Glc structures. The enzyme is activated by divalent cations and has a maximum of activity at pH 5. It is unable to transfer fucose to sialylated glycoproteins, 6-alpha-sialyllactose or 3-alpha-sialyllactose. As suggested by its precipitation in the presence of antibodies raised in rabbit against a soluble human milk N-acetylglucosaminide 3-alpha-L-fucosyltransferase, these two enzymes seem to be structurally related.

Alpha-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8-32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration. These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted alpha-lactalbumin. Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of alpha-lactalbumin. Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human alpha-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50. Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.

alpha-Lactalbumin from human milk shows an heterogeneous behaviour when subjected to ion exchange chromatography with DEAE-Sephadex. Two components have been separated, showing identical patterns in the following studies: amino acid compositions, fluorescence and circular dichroism spectra, transition temperature of denaturation, antigenicity, lactose synthase specifying activity and hydrodynamic properties. After rechromatography of either peak, these two components appeared to be in equilibrium. This equilibrium varies with the temperature and the pH of chromatography. Moreover, an increase of n-alcohol concentration in the eluting buffer also induces an increase of the second protein peak eluting at higher ionic strength. These two peaks seem to be the result of some conformational change induced upon the binding of the protein to the solid anionic matrix.

Six purified glycosyltransferase (a beta-galactoside alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 2 leads to 3 sialyltransferase, an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 1 leads to 2 fucosyltransferase, a beta-N-acetylglucosaminide alpha 1 leads to 3 fucosyltransferase, and a (fucosyl alpha 1 leads to 2) galactoside alpha 1 leads to 3 N-acetyl-galactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man, and antifreeze glycoprotein, which contains oligosaccarides with the structure, Gal beta 1 leads to 3GalNAc alph 1 leads O-Thr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the substrate specificities of the individual enzymes dictate the structures that can be synthesized and the pathways by which they may be formed. The actions of a number of the transferasesare mutually exclusive, thereby prohibiting the formation of theoretically possible oligosaccharide structures. Oligosaccharides with the terminal sequence NeuAc alpha 2 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc cannot be formed because the prior incorporation of sialic acid by the sialyltransferases yields products that are not acceptor substrates for the fucosyltransferases, and vice versa. Synthesis of other products requires that the enzymes act sequentially in a specific order. The structures NeuAc alpha 2 leads to 6(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, Fuc alpha 1 leads to 2Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc, GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc can only be synthesized if the fucosyl alpha 1 leads to 2 galactose linkage is formed first. Synthesis of the pentasaccharide sequences GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc requires that the N-acetylgalactosaminyltransferase act last on the former structure and that the alpha 1 leads to 3 fucosyltransferase act last on the latter. In those instances where a product can be formed by one of two possible pathways, the comparisons of reaction rates indicate that one pathway is usually preferred...