Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

Comparative testing of disinfectant and antiseptic products using proposed European suspension testing methods

The development of standard suspension test methods for disinfectants and antiseptics for adoption in Europe is described. Evaluation of a range of disinfectant agents and products currently used in the UK under conditions as proposed for these tests indicates that the majority of products diluted in water of standard hardness showed satisfactory activity producing a 4·5–5 log reduction in viable count within 5 min against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Proteus mirabilis and Candida albicans in the absence and presence of 1% albumin. All the products, when diluted with distilled water, produced greater than 5 log reduction in 60 min.     

Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

The Fc portion of intact IgG blocks stimulation of human PBMC by anti-T3

The means by which normal human serum inhibits the activation of PBMC by OKT3 has been investigated. The Fc portion of intact IgG is shown to be the major inhibitor in human serum of this OKT3-induced stimulation. Furthermore, inhibition by IgG subclasses correlated with their ability to bind to the monocyte Fc receptor, i.e., IgG1 and IgG3 produced greater inhibition than IgG2 and IgG4, and this inhibition was competitive. In contrast, hypogammaglobulinemic serum, IgA, IgM, and F(ab')2 of IgG were not inhibitory relative to intact IgG or Fc of IgG. Because of the similarities between T3 and the idiotype-defined T cell receptor for antigen, these investigations suggest that IgG might modulate the interactions between the T cell recognition complex and anti-idiotype antibody, thus regulating the idiotype network.     

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.