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PubMed1992

Crystallization of biologically active hemagglutinin-neuraminidase glycoprotein dimers proteolytically cleaved from human parainfluenza virus type 1.

We isolated, purified, and characterized the hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 1, with the ultimate goal of producing crystals suitable for three-dimensional X-ray structure analysis. Pronase was used to cleave the globular head of the HN molecule directly from virus particles, forming HN monomers and dimers. The purified dimers retained neuraminidase and hemadsorption activity and were recognized by 14 anti-HN monoclonal antibodies, demonstrating intact HN antigenic structure and function. N-terminal sequence analysis of the dimers showed that cleavage had occurred at amino acid 136 or 137, freeing the C-terminal 438 or 439 amino acids. On electron micrography, the dimer appeared as two box-shaped structures, each approximately 5 by 5 nm. When the purified HN dimers were crystallized in hanging drops by vapor diffusion against 20% polyethylene glycol 3350, they formed both rectangular plates and needlelike crystals. The rectangular crystals diffracted X-rays, indicating an ordered atomic structure. However, the resolution was approximately 10 A (1 nm), insufficient for three-dimensional structural analysis. Experiments to improve the resolution by increasing the size and quality of the crystals are in progress.

Amino Acid Sequence↗

PubMed1994

Analysis of the primary T-cell response to Sendai virus infection in C57BL/6 mice: CD4+ T-cell recognition is directed predominantly to the hemagglutinin-neuraminidase glycoprotein.

Sendai virus infection of C57BL/6 mice elicits a strong CD4+ and CD8+ T-cell response in the respiratory tract. To investigate the specificity of the CD4+ T-cell response, a panel of hybridomas was generated from cells recovered from the respiratory tracts of infected mice. Using vaccinia virus recombinants expressing individual Sendai virus proteins, we found that the majority of these hybridomas (34 of 37) were specific for the hemagglutinin-neuraminidase (HN) glycoprotein. The hybridomas were then analyzed for reactivity to a set of overlapping peptides spanning the entire length of the hemagglutinin-neuraminidase glycoprotein. At least five H-2 I-Ab-restricted epitopes were defined in HN. The strong bias toward recognition of class II epitopes derived from a single viral protein contrasts with T-cell recognition of epitopes of several proteins in influenza A virus as found previously by others.

Amino Acid Sequence↗

PubMed2003

Fusion deficiency induced by mutations at the dimer interface in the Newcastle disease virus hemagglutinin-neuraminidase is due to a temperature-dependent defect in receptor binding.

The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37 degrees C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.

Animals↗

PubMed2003

Triggering of human parainfluenza virus 3 fusion protein (F) by the hemagglutinin-neuraminidase (HN) protein: an HN mutation diminishes the rate of F activation and fusion.

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

Animals↗

PubMed2004

Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is an important determinant of its virulence. We investigated the role of each of the four functional N-linked glycosylation sites (G1 to G4) of the HN glycoprotein of NDV on its pathogenicity. The N-linked glycosylation sites G1 to G4 at residues 119, 341, 433, and 481, respectively, of a moderately pathogenic NDV strain Beaudette C (BC) were eliminated individually by site-directed mutagenesis on a full-length cDNA clone of BC. A double mutant (G12) was also created by eliminating the first and second glycosylation sites at residues 119 and 341, respectively. Infectious virus was recovered from each of the cDNA clones of the HN glycoprotein mutants, employing a reverse genetics technique. There was a greater delay in the replication of G4 and G12 mutant viruses than in the parental virus. Loss of glycosylation does not affect the receptor recognition by HN glycoprotein of NDV. The neuraminidase activity of G4 and G12 mutant viruses and the fusogenicity of the G4 mutant virus were significantly lower than those of the parental virus. The fusogenicity of the double mutant virus (G12) was significantly higher than that of the parental virus. Cell surface expression of the G4 virus HN was significantly lower than that of the parental virus. The antigenic reactivities of the mutants to a panel of monoclonal antibodies against the HN protein indicated that removal of glycosylation from the HN protein increased (G1, G3, and G12) or decreased (G2 and G4) the formation of antigenic sites, depending on their location. In standard tests to assess virulence in chickens, all of the glycosylation mutants were less virulent than the parental BC virus, but the G4 and G12 mutants were the least virulent.

Antibodies, Monoclonal↗

PubMed2004

Mutated form of the Newcastle disease virus hemagglutinin-neuraminidase interacts with the homologous fusion protein despite deficiencies in both receptor recognition and fusion promotion.

The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segments from HN proteins derived from two different NDV strains. These findings suggest that the I175E substitution converts HN to an F-interactive form, but it is one for which receptor binding is still required for fusion promotion. The data also indicate that the integrity of the HN dimer interface is critical to its receptor recognition activity.

Amino Acid Sequence↗

PubMed2004

Biological significance of the second receptor binding site of Newcastle disease virus hemagglutinin-neuraminidase protein.

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-alpha(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.

Binding Sites↗

PubMed2004

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.

Animals↗

PubMed2003

Functional analysis of individual oligosaccharide chains of Sendai virus hemagglutinin-neuraminidase protein.

The roles of N-linked glycosylation in the intracellular transport and biological activities of the Sendai virus hemagglutinin-neuraminidase (HN) protein were studied. The protein contains four potential N-glycosylation sites: N77, N448, N499, and N511. By site-directed mutagenesis of these positions, the mature protein contained three N-linked oligosaccharides attached to N77, N499, and N511. The role of each added oligosaccharide in the structure and functions of the protein was identified by characterization of surface expression, hemadsorption, and neuraminidase activities of the corresponding mutant proteins. Elimination of the sites of N499 and N511 had the most detrimental effect, decreasing surface expression and hemadsorption. Elimination of the sites of N77 and N448 had similar but weaker effects. Mutants missing the sites of N499 and N511 were not able to induce syncytia formation in cells expressing mutant HN proteins and the fusion protein. Therefore, the N-linked oligosaccharides attached to N499 and N511 were important for intracellular transport and for the fusion promotion.

Animals↗

PubMed1993

Structure and function of a membrane anchor-less form of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses is anchored in the virion membrane near its amino terminus, protruding from the virion surface to mediate attachment to cellular receptors. Solubilization of HN spikes can be achieved by treatment of virions with detergent and high salt concentrations. When the solubilized HN protein from the Australia-Victoria (AV) isolate of the virus is incubated at 37 degrees C, a chymotrypsin-sensitive site between residues 112 and 113 is exposed. A chymotrypsin-cleaved soluble form of the protein, named CT-HN, has been prepared using this approach. It is membrane anchor-less, due to removal of a 14-kDa fragment from the NH2 terminus of HN. It retains all potential glycosylation sites and cysteines present in the ectodomain of the native protein. It migrates in nonreducing gels and sediments in sucrose gradients at the rate expected for homodimeric HN. The latter is also consistent with our demonstration by site-directed mutagenesis that cysteine residues at positions 6 and 123, respectively, mediate disulfide-linked homotetramer and homodimer formation. CT-HN retains almost total antigenicity, suggesting that it is conformationally very similar to the intact molecule, as well as receptor recognition function and, at low pH, neuraminidase activity. It should prove to be a useful tool for further studies of the structure and function of this important viral glycoprotein.

Amino Acid Sequence↗

PubMed1963

SIALIDASE (NEURAMINIDASE) OF CORYNEBACTERIUM DIPHTHERIAE.

Warren, Leonard (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.) and C. W. Spearing. Sialidase (neuraminidase) of Corynebacterium diphtheriae. J. Bacteriol. 86:950-955. 1963.-The characteristics of a sialidase produced by Corynebacterium diphtheriae were studied. The enzyme was partially purified from preparations of diphtheria toxin on a column of Sephadex G-75. By this means the lethal factor of diphtheria toxin was separated, in part, from the sialidase activity. There appeared to be a close immunological relationship between the sialidases of C. diphtheriae and clostridia, since a preparation of diphtheria antitoxin was as effective an inhibitor of diphtheria sialidase as of the sialidase of three species of clostridia. Conversely, antitoxin to clostridia inhibited diphtheria sialidase. Diphtheria antitoxin was essentially inactive toward influenza virus sialidase, and was completely inactive against purified sialidase of Vibrio cholerae. Removal of sialic acid from the proteins in a preparation of diphtheria antitoxin did not alter the inhibitory activity of the antitoxin against diphtheria sialidase. The enzyme operated optimally at pH 5.5 and did not require calcium ions for activity. The substrate specificity of diphtheria sialidase appears to be the same as that of other previously described sialidases.

Corynebacterium diphtheriae↗

PubMed2000

Neuraminidase inhibitors for preventing and treating influenza in healthy adults.

OBJECTIVES: Influenza is an acute respiratory infection associated with raised temperature, headache, muscle ache and cough. The objective of this review was to assess the effects of neuraminidase inhibitors (NIs) in preventing cases of influenza and shortening or reducing the severity of influenza in healthy adults. A further objective was to estimate the frequency of adverse effects associated with NI administration. SEARCH STRATEGY: We searched Medline, the Cochrane Acute Respiratory Infections Group trials register, the Cochrane Controlled Trials Register (CCTR), manufacturers' databases, Embase (1991 to 1998) and reference lists of articles in May 1999. We also contacted manufacturers, researchers in the field, and authors of studies evaluated in the review. SELECTION CRITERIA: Randomised or quasi-randomised placebo-controlled studies of NIs in healthy adults. Studies assessing protection or treatment from exposure to naturally occurring and experimental influenza were considered. The main outcomes were numbers and/or severity of influenza cases and the number and seriousness of adverse effects. DATA COLLECTION AND ANALYSIS: Two reviewers applied the inclusion criteria to the retrieved studies, assessed trial quality and extracted data. MAIN RESULTS: Eight trials with 1180 adults were included. Overall the methodological quality of the studies appeared to be good. As a preventive measure, NIs when compared to placebo were 74% effective (95% confidence interval 50% to 87%) in preventing naturally occurring cases of clinically defined influenza, and 60% effective (95% confidence interval 76% to 33%) in preventing cases of laboratory confirmed influenza. As a treatment, NIs shorten the duration of symptoms by one day - weighted mean difference 1 (95% confidence interval -1.3 to -0.6). The time gained in returning to normal activities is half a day - weighted mean difference -0.5 ( 95% confidence interval -1.1 to -0.1) for laboratory cases of influenza. The adverse event profile (local nasal irritation) of Zanamivir appears no better than placebo - odds ratio 1.19 ( 95% confidence interval 0.39 to 3.62). Compared with rimantadine in a preventive role, Oseltamivir has a significantly lower incidence of adverse effects and significantly higher incidence of nausea. For treatment, the adverse event profile shows that gastrointestinal symptoms are significantly worse in NIs than placebo - Peto odds ratio 2.32 ( 95% confidence interval 1.55 to 3.47). REVIEWER'S CONCLUSIONS: NIs are effective for the prevention and treatment of influenza. Overall NIs are safe, although Oseltamivir causes significant nausea.

Adult↗

PubMed2006

Neuraminidase inhibitors for preventing and treating influenza in healthy adults.

BACKGROUND: Neuraminidase inhibitors (NI) are recommended for use against influenza and its complications in interpandemic years and in a pandemic. OBJECTIVES: To assess the effects of NIs in preventing or ameliorating influenza, its transmission and its complications in healthy adults and to estimate the frequency of adverse effects. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 3, 2005), MEDLINE (2004 to September, Week 4 2005), EMBASE (2003 to June 2005) and contacted manufacturers, researchers in the field, and authors of studies evaluated in the review. SELECTION CRITERIA: Randomised or quasi-randomised placebo-controlled studies of NIs in healthy adults exposed to naturally occurring influenza. DATA COLLECTION AND ANALYSIS: Two authors applied inclusion criteria, assessed trial quality and extracted data. We structured the comparisons into prophylaxis, treatment and adverse events with further subdivision by outcome and dose. MAIN RESULTS: We identified four prophylaxis, 13 treatment and four post-exposure prophylaxis (PEP) trials. In prophylaxis compared to placebo, NIs have no effect against influenza-like illnesses (ILI) (relative risk (RR) 1.28, 95% confidence interval (CI) 0.45 to 3.66 for oral oseltamivir 75 mg daily; RR 1.51, 95% CI 0.77 to 2.95 for inhaled zanamivir 10 mg daily). The efficacy of oral oseltamivir 75 mg daily against symptomatic influenza is 61% (RR 0.39, 95% CI 0.18 to 0.85), or 73% (RR 0.27, 95% CI 0.11 to 0.67) at 150 mg daily. Inhaled zanamivir 10 mg daily is 62% efficacious (RR 0.38, 95% CI 0.17 to 0.85). Neither NI has a significant effect on asymptomatic influenza. Oseltamivir induces nausea (odds ratio (OR) 1.79, 95% CI 1.10 to 2.93). Oseltamivir for PEP has an efficacy of 58.5% (15.6% to 79.6) for households and of 68% (34.9 to 84.2%) to 89% in contacts of index cases. Zanamivir has similar performance. The hazard ratios for time to alleviation of influenza symptoms were in favour of the treated group 1.33 (1.29 to 1.37) for zanamivir and 1.30 (1.13 to 1.50) for oseltamivir. Viral nasal titres were significantly diminished by both NIs. Oseltamivir 150 mg daily prevented lower respiratory tract complications (OR 0.32, 95% CI 0.18 to 0.57). We could find no comparative data on the effects of oseltamivir on avian influenza. AUTHORS' CONCLUSIONS: Because of their low effectiveness, NIs should not be used in routine seasonal influenza control. In a serious epidemic or pandemic, NIs should be used with other public health measures. We are unsure of the generalisability of our conclusions from seasonal to pandemic or avian influenza.

Acetamides↗

PubMed1977

Isolated acid neuraminidase deficiency: a distinct lysosomal storage disease.

An 8-month-old female presented with coarse facies and hepatosplenomegaly at birth. Growth proceeded at an accelerated rate and mental development was normal. A pattern of dysostosis multiplex developed radiographically. Cytoplasmic inclusions consistent with lysosomal storage disease were demonstrated by electron microscopy in bone marrow, liver, and cartilage cells and in cultured skin fibroblasts. Assays of the fibroblasts revealed a specific deficiency of acid neuraminidase and 6-fold increase in intracellular bound sialic acid. An unidentified macromolecular compound rich in sialic acid was excreted in excessive amounts in the urine. The phenotype suggests defective degradation primarily of glycoproteins and possibly to a lesser extent of keratan sulfate and gangliosides.

Bone Marrow↗

PubMed2005

One-bead-one-inhibitor-one-substrate screening of neuraminidase activity.

Given the eminent threat of a 21st century flu pandemic, the search for novel antiviral compounds is an increasingly important area of research. Recent developments in antiviral research have established the viability of targeting viral neuraminidase (NA), an enzyme that cleaves sialic acid from the cell-surface-mediating passage of the virus in the respiratory tract. N-acetyl neuraminic acid (NeuAc) is the substrate for NA, and analogues of this core structure have been commercialized as antiviral therapeutics. Recent studies have established that this system is well suited for combinatorial approaches to drug discovery. An important step in the process is to develop solid-phase screening technologies. The feasibility of performing competitive solid-phase NA assays is reported herein. Initially, a fluorogenic NeuAc substrate was immobilized on solid support, and the ability of three NAs (Clostridium perfringens, Salmonella typhimurium, and Vibrio cholerae) to cleave the substrate was shown to be analogous to solution-phase assays. The solid support was then bifunctionalized with the fluorogenic NeuAc substrate and one of two known inhibitors (DANA and Zanamivir). The ability of NA to cleave NeuAc from the solid support when simultaneously presented with an inhibitor was shown to be enzyme dependent. As expected, simultaneous presentation of NeuAc and DANA, a nonspecific inhibitor, led to diminished activity for all three enzymes tested. In contrast, dual presentation of NeuAc and the selective inhibitor Zanamivir only showed significant activity against Vibrio cholerae.

Antiviral Agents↗

PubMed1992

Glycosidase inhibitors (castanospermine and swainsonine) and neuraminidase inhibit pokeweed mitogen-induced B cell maturation.

Castanospermine (CSP), an inhibitor of alpha-glucosidase, enhanced immunoglobulin (Ig) release in a Staphylococcus aureus Cowan I (SAC)-induced lymphocyte culture (Scand. J. Immunol. 1990. 32: 529). In a pokeweed mitogen (PWM)-human lymphocyte culture, unlike the SAC-stimulated system, CSP strongly decreased the number of IgG-, IgA- and IgM-secreting cells as well as that of Ig-bearing cells. Peripheral blood lymphocytes treated with swainsonine, a mannosidase II inhibitor, or with neuraminidase also showed a reduced response to PWM. In cross-culture experiments, only a mixture of B cells pretreated with either agent and untreated T cells showed such a suppressive effect. Adhesion was decreased between B cells treated with either agent and untreated T cells, but not between treated T cells and untreated B cells. These results demonstrate that a certain alteration in B cell membrane oligosaccharides inhibited the T cell-B cell adhesion in the PWM culture, leading to an arrest of B cell maturation. Considering that these inhibitors eventually prevent terminal sialic acid addition, the present study provides evidence that sialic acids on B cell surface oligosaccharides play a biological role in the T cell-B cell interaction.

B-Lymphocytes↗

PubMed1992

Improvement of BF typing and of BF F subtyping after neuraminidase treatment in the unconverted and converted factor B.

When neuraminidase-treated sera are analyzed by agarose gel isoelectric focusing, the factor B (BF) banding pattern is reduced to predominantly one major band without cathodically positioned bands. This not only makes unequivocal typing of BF allotypes possible but also the reliable distinction of all BF F subtype phenotypes with delimitation of "BF F subtype variants". With this new method, serum aging affects the BF determination to a lesser extent than when applying methods that separate native sera. We show that sialylation is not responsible for the BF F subtype polymorphism. All of the investigated BF allotype bands, including those characteristic of the subtypes, show functional hemolytic activity. The banding pattern after removal of neuraminic acid residues ranges from pH 6.8 to 7.3 for factor B, from pH 5.3 to 5.9 for the Ba fragment, and from pH 8.2 to 8.7 for the Bb fragment. The protein structure of factor B is also discussed. Eliminating the superimposition of bands in different BF allotypes, as demonstrated by these methods, proved to be necessary for the detection of hypomorphic BF gene products (BF QL), which are expressed by assumed BF*Q0 alleles in heterozygous genotypes. This allows investigation of BF*Q0 alleles on a protein level, which complements molecular genetic approaches.

Blood Preservation↗

PubMed1974

Neuraminidase-mediated augmentation of in vitro immune response of patients with solid tumors.

Host blood lymphocytes undergo accentuated blastic transformation when cultured with tumor cells pretreated with neuraminidase. The effect has been observed in 38 patients with such common solid tumors as bronchus carcinoma, skin melanoma, hypernephroma, or adenocarcinoma of the breast, lung, colon, or rectum. Individual response varied but often exceeded response to allogeneic cells. Three patients with glioblastoma of the brain did not respond. Lymphoblastic transformation was not observed in three of four cultures containing benign tumor or in any cultures containing normal tissue analogues of the malignant tumors. A factor in host blood serum inhibiting lymphoblastic transformation correlated to abnormal elevation of serum-bound sialic acid. This blocking factor differed in specificity from enhancing antibody or serum blocking complexes described by other investigators. Blocking effects were observed when the tumor-cell type of a serum donor differed from the cell type of the culture test tumor. Serum with abnormal elevation of bound sialate from a cancerfree human also non-specifically blocked host response to tumor. The blocking effect could be eliminated by partial enzymatic removal of bound sialic acid from serum glycoproteins.

Adenocarcinoma↗