Structural basis of a conserved idiotope expressed by an autoreactive human B cell lymphoma. Evidence that a VH CDR3 mutation alters idiotypy and specificity

Our laboratory has previously investigated the relationship of autoimmune disease and B cell neoplasia in a patient with a diffuse, well differentiated splenic B cell lymphoma and associated autoimmune hemolysis due to an anti-Pr2 antibody. EBV-immortalized B cell clones, established from this lymphoma, were shown to secrete the same pathologic anti-Pr2 antibody. The antiidiotypic mAb, RI.1, defined a private Id (IdRI.1) of the anti-Pr2 antibody that was related to the Ag-binding site and was expressed by both the lymphoma and derived cell lines. This unique Id was expressed by the majority of splenic tumor B cells and also was conserved over a period of 4 yr. In this report, the structural basis of IdRI.1 expression was investigated by analysis of Id- variants isolated by flow microfluorimetry using RI.1. Six Id- cell lines that secrete IgM kappa but lack Pr2 specificity were generated from an Id+ cell line, LS2. These lines were shown to be related to LS2 and the lymphoma by karyotype and by restriction fragment analysis of Ig gene rearrangements. Shared and unshared nucleotide substitutions in the VH and VL regions of the six independent clones were used to construct a genealogic tree relating the Id- clonal members to a common Id+ precursor. The tree illustrates that the base changes occurred sequentially, suggesting that they were introduced by somatic point mutation. Only one VH CDR3 bp difference from the LS2 nucleic acid sequence is common to all Id- sequences, resulting in an amino acid substitution of cysteine 108 to tyrosine. Taken together, these findings suggest that both the expression of IdRI.1 and Ag binding are affected by a single mutation localized to the D region of the anti-Pr2 antibody.     

Systematic mutation of bacteriophage T4 lysozyme

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).     

Effect of medium on growth and subsequent survival,after freeze-drying,ofLactobacillus delbrueckii subsp.bulgaricus

Summary Growth ofLactobacillus delbrueckii subsp.bulgaricus, grown at a constant pH (5.7) in papain-or pepsin-treated whey was superior to that in whey or whey permeate, but inferior to growth in milk; it was not significantly different from that found in a 1:1 whey: milk medium. When 2% sodium citrate was added to the fermented medium, the cell recovery levels (59–75%) upon centrifugation were not significantly different in papain-treated media compared to untreated substrates. Cultures obtained from papain-treated milk or whey showed similar mortality levels following freeze-drying (99%) to those grown on untreated milk or whey. Addition of Tween 80 to the growth medium improved survival rate by a factor of ten.     

Three-dimensional model of cancer and in vitro metastasis: application to non-Hodgkin malignant lymphoma

In vitro cancer studies require models more appropriate than the standard monolayer cultures of tumoral cell lines. This report describes the production of an in vitro three-dimensional rebuilt tumor using a non-hodgkin malignant lymphoma. The model exploits the relationship between angiogenesis and cancer formation by employing both tumor cells and fusiform cells derived from an angioma. The significance of this model, which has also been used with malignant melanoma cells, is that the rebuilt tumor, when placed in culture, produces many tumorous nodules which are fixed to a sub-layer of fusiform cells and newly-secreted matrix. These are, in effect, in vitro metastases. The ultrastructural aspect of this neomatrix indicates its proteoglycan nature. The micro-environment formed by the vascular cells and matrix appears to be critical for the production of metastases.     

Metabolic effect at six and twelve months of cyproterone acetate (2 mg) combined with ethinyl estradiol (35 micrograms) in 31 patients

The metabolic effects of cyproterone acetate (2 mg) combined with a new dose level of ethinyl estradiol (35 micrograms) were studied over a one-year period in 31 patients presenting moderate clinical hyperandrogenism. The following tests were performed before treatment, at 6 and 12 months, an oral glucose tolerance test (OGTT) with measurement of insulinemia, total cholesterol, HDL cholesterol and the LDL + VLDL/HDL ratio, A1 and B apoproteins. At six months and at one year of treatment, the weight and body mass index (kg/m2) were not modified. Glucose tolerance and insulinemia had not changed significantly at one year. Lipid test results showed an increase in triglycerides, as well as in total and HDL cholesterol levels. However, the LDL + VLDL/HDL and A1/B apoprotein ratios did not change during the study. We conclude from these results that the new combination does not have any adverse effects on glucose tolerance and has a predominantly estrogenic effect on lipid parameters, characterized by increases in total cholesterol, HDL cholesterol and triglycerides.     

Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate

The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.     

Proposal for Naming Host Cell-Derived Inserts in Retrovirus Genomes

We propose a system for naming inserted sequences in transforming retroviruses (i.e., onc genes), based on using trivial names derived from a prototype strain of virus.     

Differential Transmitter Release from Nerve Terminals Isolated from Basal Ganglia and Substantia Nigra

Abstract: The K⁺-induced release of amino acids and dopamine from synaptosomes of basal ganglia and substantia nigra of sheep was studied. K⁺ (56 mM) caused an increase in the release of GABA from caudate, putamen, globus pallidus, and substantia nigra, the increased release being 227, 171, 198, and 366%, respectively, compared with samples incubated without stimulation. The release of glutamate was also increased by 56 mM-K⁺ (136–183%) from all regions except the globus pallidus, and a significant release of aspartate was only seen in response to K⁺ stimulation of synaptosomes from putamen (50%). Veratrine (75 μM) also stimulated a similar pattern of amino acid release from these regions. Regional correlation was shown between the presence of an uptake system for an amino acid and its evoked release. [¹⁴C]Dopamine formed from L-[U-¹⁴C]tyrosine was released only from caudate and putamen synaptosomes by K⁺ stimulation, the increases being 105% and 74%, respectively. Synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine occurred only in synaptosomes prepared from these two regions and was not detected in synaptosomes from substantia nigra or globus pallidus although whole-tissue homogenates of substantia nigra were able to synthesise dopamine.     

Influence of growth environment on the phosphoenolpyruvate: Glucose phosphotransferase activities of Escherichia coli and Klebsiella aerogenes: A comparative study

A consistent difference was found between glucose-limited cultures of Escherichia coli and Klebsiella aerogenes strains in the manner which their apparent cellular content of glucose: phosphoenolpyruvate phosphotransferase (glucose-PTS) varied with growth rate. With the former strains, activity increased as a function of growth rate; in the latter it decreased. However, under glucose-sufficient conditions (potassium-or ammonia-limitation) both species behaved similarly; the glucose-PTS activity was lower and bore no obvious relationship to the rate of glucose consumption expressed by the growing culture. These results are discussed in relation to the role of glucose as a regulator of glucose-PTS synthesis, and to the likely contribution which the glucose-PTS makes to the overall rate of glucose uptake, particularly by cells growing in glucose-sufficient environments.Abbreviation Glucose-PTS phosphoenolpyruvate phosphotransferase From May to November 1978 on study leave in the University of Amsterdam     

Radiosensitization of hypoxic mammalian cells in vitro by some 5-substituted-4-nitroimidazoles

The efficiencies of various 5-substituted-4-nitroimidazoles as radiation sensitizers have been determined in hypoxic Chinese hamster cells irradiated in vitro. Compared with published data on the sensitizing properties of substituted 2-nitro- and 5-nitroimidazoles, some of the 4-nitro derivatives show unusually high sensitizing efficiencies defined as the concentrations required to give an enhancement ratio of 1.6. The equilibrium one-electron reduction potentials of the compounds (E17) were measured by a pulse radiolysis technique and the results show that although sensitizing efficiencies are unexpectedly high, based on considerations of electron affinity, they still increase with increasing values of E17. Enhancement ratios were determined in two V79 cell lines for combinations of one of these compounds (a 4-nitroimidazole containing the group SO2.O.phenyl in the 5-position, NSC 38087) with various concentrations of misonidazole. The sensitization observed suggests that the two compounds may be operating by different mechanisms.