Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures.

Major heat shock proteins, such as the Escherichia coli DnaK protein, not only are required for cell growth after heat shock but seem to possess important functions in cellular metabolism at normal growth temperatures as well. E. coli delta dnaK52 mutants have severe cellular defects at 30 degrees C, one of which is in cell division (B. Bukau and G. C. Walker, J. Bacteriol, 171:2337-2346, 1989). Here we show that at 30 degrees C, delta dnaK52 mutants have defects in chromosome segregation and in maintenance of low-copy-number plasmids. Fluorescence microscopic analysis revealed that chromosomes were frequently lacking at peripheries of cell filaments of delta dnaK52 mutants and clustered at other locations. In other parts of the cell filaments, chromosomes were apparently normally distributed and they were also present in most of the small cells found in populations of delta dnaK52 cells. These defects might be at the level of DNA replication, since delta dnaK52 mutants have a threshold lower rate of DNA synthesis than wild-type cells. Chromosome segregation defects of delta dnaK52 mutants were also observed in an rnh dnaA mutant background, in which initiation of DNA replication is DnaA-oriC independent. We also found that low-copy-number P1 miniplasmids could not be stably maintained in delta dnaK52 mutants at 30 degrees C. delta par P1 miniplasmids that carry the P1-encoded rep functions required for their replication but lack the P1-encoded par functions required for faithful partitioning of the plasmids during cell division were also unstable in delta dnaK52 mutants. Taken together, our results indicate important, although not absolutely essential, functions for DnaK at 30 degrees C in one or more processes necessary for correct replication and/or partitioning of chromosomes and P1 miniplasmids. Furthermore, we found that P1 miniplasmids were also highly unstable in dnaJ259 mutants, indicating a role for the DnaJ heat shock protein in maintenance of these plasmids.     

Heme methyl 1H chemical shifts as structural parameters in some low-spin ferriheme proteins

 The different paramagnetic shifts of the four methyl groups in ferriheme proteins have been described as being due to the effect of the axial ligand nodal plane orientation. An equation, heuristically found and theoretically explained, describing the relation between contact and pseudocontact shifts and the position of the axial ligand(s) has been derived for bis-histidine ferriheme proteins and for cyanide-histidine ferriheme proteins. The values of the heuristic parameters contained in the equations were found by fitting the shifts of bovine cytochrome b ₅ and several bis-histidine cytochromes c ₃ and histidine-cyanide systems. The agreement between the observed and the calculated shifts was found to be good. Therefore, by taking advantage of this study, information on the position of the axial ligands, that can be used as a constraint for structure determination, can be obtained from the shifts of the methyl protons. Received: 13 April 1999 / Accepted: 4 June 1999     

Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif

Using a combination of solid phase synthesis for the preparation of N-substituted-N-acylglycines 7 followed by solution-phase ring transformation of trifluoromethylacyl munchnone intermediate 8, a library of 200 trisubstituted-5-trifluoromethylketo (TFMK) imidazoles 9 was prepared. In a sublibrary, bromoacetate resin 4 was treated with 5 amines in parallel to give N-substituted glycines 5 followed by acylation with 12 acid chlorides to provide, upon cleavage from the resin, 60 individual N-substituted-N-acylglycines 7. The glycines 7 were converted to munchnones 8 by treatment with trifluoroacetic anhydride followed by reaction with benzamidine to give trisubstituted-5-TFMK-imidazoles 9. The structural content of the library was analyzed using PlateView of the LCMS results, and individual members were isolated by automated preparative LCMS.     

Resolution of the membrane domain of bovine complex I into subcomplexes: implications for the structural organization of the enzyme

Complex I (NADH:ubiquinone oxidoreductase) purified from bovine heart mitochondria was treated with the detergent N, N-dimethyldodecylamine N-oxide (LDAO). The enzyme dissociated into two known subcomplexes, Ialpha and Ibeta, containing mostly hydrophilic and hydrophobic subunits, and a previously undetected fragment referred to as Igamma. Subcomplex Igamma contains the hydrophobic subunits ND1, ND2, ND3, and ND4L which are encoded in the mitochondrial genome, and the nuclear-encoded subunit KFYI. During size-exclusion chromatography in the presence of LDAO, subcomplex Ialpha lost several subunits and formed another characterized subcomplex known as Ilambda. Similarly, subcomplex Ibeta dissociated into two smaller subcomplexes, one of which contains the hydrophobic subunits ND4 and ND5; subcomplex Igamma released a fragment containing ND1 and ND2. These results suggest that in the intact complex subunits ND1 and ND2 are likely to be in a different region of the membrane domain than subunits ND4 and ND5. The compositions of the various subcomplexes and fragments of complex I provide an organization of the subunits of the enzyme in the framework of the known low resolution structure of the enzyme.     

Dimethylsulfoxide oxidizes glutathione in vitro and in human erythrocytes: kinetic analysis by 1H NMR

The interaction of dimethylsulfoxide (Me2SO) with glutathione was investigated under non-equilibrium conditions in solution using 1H NMR and in intact erythrocytes using 1H spin-echo NMR. In solution the reaction was observed to follow second-order kinetics (Rate = k1[glutathione][Me2SO]) at 300 K pH 7.4, k(sol) = 4.7 x 10(-5)mol(-1)L(1)s(-1). In intact erythrocytes the rate constant for the cellular environment, k(cell), was found to be slightly larger at 8.1 x 10(-5)mol(-1)L(1)s(-1). Furthermore, the reaction of Me2SO with erythrocyte glutathione showed a biphasic dependence on the Me2SO concentration, with little oxidation of glutathione occurring until the Me2SO concentration exceeded 0.5 molL(-1). The results suggest that at lower concentrations, Me2SO can be effectively removed, most probably by reaction with glutathione, which is regenerated by glutathione reductase, although preferential reaction with other cellular components (e.g., membrane or cellular thiols) cannot be ruled out. Thus the concentrations of Me2SO that are commonly used in cryopreservation of mammalian cells ( approximately 1.4 molL(-1)) can cause oxidation of intracellular glutathione.     

Lack of Viral Escape and Defective In Vivo Activation of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes in Rapidly Progressive Infection

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.     

Soil pCO2, soil respiration,and root activity in CO2-fumigated and nitrogen-fertilized ponderosa pine

The purpose of this paper is to describe the effects of CO₂ and N treatments on soil pCO₂, calculated CO₂ efflux, root biomass and soil carbon in open-top chambers planted with Pinus ponderosa seedlings. Based upon the literature, it was hypothesized that both elevated CO₂ and N would cause increased root biomass which would in turn cause increases in both total soil CO₂ efflux and microbial respiration. This hypothesis was only supported in part: both CO₂ and N treatments caused significant increases in root biomass, soil pCO₂, and calculated CO₂ efflux, but there were no differences in soil microbial respiration measured in the laboratory. Both correlative and quantitative comparisons of CO₂ efflux rates indicated that microbial respiration contributes little to total soil CO₂ efflux in the field. Measurements of soil pCO₂ and calculated CO₂ efflux provided inexpensive, non-invasive, and relatively sensitive indices of belowground response to CO₂ and N treatments.