Subclass specificity of the Fc receptor for human IgG on K562

The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).     

The interaction of CD2 with its LFA-3 ligand expressed by autologous erythrocytes results in enhancement of B cell responses

The addition of autologous erythrocytes to unfractionated human mononuclear cell cultures results in enhancement of B cell responses to antigens and mitogens. This costimulating effect of red cells is abrogated by their preincubation with anti-LFA-3 monoclonal antibody. Preincubation of mononuclear cells with anti-CD2 monoclonal antibodies (anti-Leu 5b, OKT11, used singly) has a down-regulating effect on B cell activation and no enhancement of B cell responses is seen when red cells are added to anti-CD2-treated cultures. These results demonstrate a functional effect on B cells of the interaction between the CD2 molecule on T lymphocytes and its natural ligand, LFA-3. The precise mechanism by which this costimulating effect on B lymphocytes takes place is unclear. The study of T cell populations and T cell activation markers shows that the addition of erythrocytes causes a small but reproducible increase in the number of cells expressing the IL-2 receptor and the addition of IL-2 enhances the response of mononuclear cells to antigenic stimulation in the presence of erythrocytes. However, the supernatants of mononuclear cell cultures stimulated with pokeweed mitogen in the presence of autologous erythrocytes show decreased levels of IL-2, compared to supernatants of cells stimulated with pokeweed mitogen alone. The same supernatants show increased levels of interferon-gamma, but the addition of this lymphokine to cultures stimulated with pokeweed mitogen has no potentiating effect. It is possible that the effect of erythrocytes is mediated by other growth and/or differentiation factors, and additional studies will be required to clarify this point.     

Properties ofAzotobacter vinelandii in phosphate-limited batch cultures

Batch cultures ofA. vinelandii in ammonium phosphate-limited and N-free phosphate-limited media were compared with control cultures (N-free phosphate-sufficient media). The effects of phosphate limitation on growth were determined by viable cells counts. Under phosphate-limitation conditions, growth inhibition and decreased viability were observed. Intracellular levels of RNA, poly-3-hydroxybutyrate, phosphate and oxygen uptake were significantly affected by phosphate limitation. When phosphate-limited cultures were examined microscopically, pleomorphism was more marked than in control cultures. Also phosphate-limited cells showed an increase in resistance to UV irradiation, mechanical disruption, desiceation and the combined action of ethylenediaminetetraacetie acid and lysozyme.     

Negative occurrence between hippocampal rhythmic slow activity and epileptiform spikes: influence of the striatum

The effects of caudate and septal stimulation on hippocampal activity were studied. Sodium penicillin was intravenously injected in order to induce a steady rate of interictal epileptic activity. Penicillin injection caused significant reduction of the rate of occurrence and duration of hippocampal rhythmic slow activity (RSA). Spontaneous RSA as well as RSA-eliciting caudate and septal stimulation induced a marked inhibition on frequency of epileptiform complexes. Lesions of the medial septal nucleus made it impossible to record RSA or to observe any sort of influence on hippocampal epileptiform activity by caudate stimulation. This result suggests that the septum is important for RSA genesis in the striato-hippocampal pathway or in the reciprocal septo-hippocampal connections.     

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary and secondary structural homologies between the HIS4 gene product of Saccharomyces cerevisiae and the hisIE and hisD gene products of Escherichia coli and Salmonella typhimurium

Summary A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of hisIE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.     

Cytometry and flow cytometry of 4',6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants

Cytometry and flow cytometry were used to study characteristics of fluorescence of the DNA-DAPI complex in nuclei released from different fresh and formaldehyde-fixed pea ( Pisum sativum L. cv. Lincoln) tissues. The two methods of isolation are compared and discussed as well as their possible use for quantitative analysis of DNA in plant tissues. With fixed tissues it is possible to obtain a number of nuclei sufficient for the flow cytometric analysis, even using small amounts of plant tissue.     

A Method for Visualizing the Acrosome by Light Microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol: acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

Computer-assisted mathematical analysis of sigmoid biological events

A program in BASIC is described which allows accurate quantificationof some numerical parameters that can be objectively correlatedto biological indexes in sigmoid biological events. Attentionwas focused on the polymerization process of actin (a muscleprotein with a mol. wt of 42 000 daltons) studied as the variationin the OD₃₆₀ index with time. The experimental points, if plotted,can be well approximated by a rational function of the typeOD₃₆₀ = f(t), which passes through the origin and can be representedgraphically by a sigmoid curve. The program was very helpfulin comparing the experimental curves and in analysing significantparameters, such as maximum velocity and asymptote, that characterizethese curves and whose interpretation would otherwise be purelysubjective. Received on July 11, 1985; accepted on January 13, 1986     

Myoglobin: cytochrome b5 interactions and the kinetic mechanism of metmyoglobin reductase

Metmyoglobin (metMb) reduction by metMb reductase from heart muscle requires cytochrome b5 as electron-transfer mediator. The existence of a metMb-ferrous cytochrome b5 complex is demonstrated by mutual perturbation of the proteins' respective electrophoretic titration curves between pH 4 and 7. The same technique shows a preferential binding of cytochrome b5 over metMb by the enzyme. The paramagnetic hyperfine shifts in the cytochrome b5 1H NMR spectrum are perturbed by metMb, indicating the formation of a specific bimolecular complex with a 1:1 stoichiometry and a binding constant estimated to be less than 10 microM. The resonances assigned to the cytochrome b5 heme 6-propionate methylene group exhibit the largest complexation shifts. Computer modeling implicates lysines 47, 50, and 98 of metMb as contact points with cytochrome b5 carboxylate residues 43, 44, 60, and heme 6-propionate. The mechanism of the enzymatic reduction establishes metMb reductase as an NADH-cytochrome b5 oxidoreductase. Cytochrome b5 is reduced at near diffusion-controlled rates by the enzyme with a turnover number of 1000 min-1 X Km for the cytochrome is 0.9 microM versus 100 microM reported for the erythrocyte enzyme. Ferrous cytochrome b5 then reduces metMb nonenzymatically with an apparent rate constant of 4.9 X 10(4) M-1 min-1 X Acetylation of metMb, which does not affect its oxygen affinity or chemical reduction, renders it a poor substrate for enzymatic reduction. This study suggests a function for the three exterior lysine residues conserved in all mammalian myoglobin sequences: they are contact points for complexation with cytochrome b5.