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Results obtained from a meiotic study of 250 pachytene cells from four normal human males are presented. G-banding patterns for pachytene bivalents, obtained using trypsin treatment, are also presented; a comparative study between G-banding patterns of pachytene bivalents and G-banding patterns of mitotic chromosomes, and a study of chromomere counting and distribution, have also been made.
A comparative immunofluorescence and light microscopical study of the three cell types of the juxtaglomerular apparatus (pure muscle cells, pure granular cells and mixed cells) was performed on the growing and maturing kidney of the rat. Mixed cells, containing contractile protein and secretory granules, are detectable on the first postnatal day in about one third of the JGAs. From the third week, the number of bivalent cells increases, while the proportion of pure muscle or pure granular cells decreases. Morphological and functional maturation, achieved by 3 to 4 weeks, is associated with increasing numbers of bivalent cells and a shift in the main site of renin production from the inner to the outer cortical zone. The divergent internal structure of JGA cell types expresses the range of varied differentiation expressed by one cell line. Pure muscle or granular cells are at the extremes of the range and mixed cells take up an intermediate position.
The mitotic Z and W sex chromosomes in Pleurodeles seem to be identical. Earlier morphological and molecular analyses of lampbrush paired chromosomes in the female meiosis showed clearly that 20% of the chromosomal length located in the middle part of the sex bivalent (bivalent IV) is heteromorphic. We investigated here the base content and composition of the DNA axes in the heteromorphic region by quantitative fluorescence imaging using various base-specific (DAPI, Hoechst 33342 and chromo-mycin A3) or base-nonspecific (ethidium bromide) fluorescent DNA probes. Our results show a significantly higher percentage of AT bases in Z than in W differential sectors. In addition the entire base content of Z appears slightly higher than that of W.
In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by the HER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amont of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185HER2 receptor.
A clinical trial was conducted with Alice/R-75 strain live attenuated intranasal influenza A/B vaccine. With double blind control 88 adult volunteers were administered 2 doses of Alice/R-75 vaccine, 93 volunteers received one dose of Alice/R-75 vaccine and one dose placebo solution and 94 subjects were administered 2 doses of placebo solution. Twenty-three other subjects received Alice strain monovalent influenza A vaccine. For comparison, data from 21 subjects who received monovalent intranasal R-75 strain influenza B in two doses is included. The vaccine was generally well tolerated. Four-fold serum hemagglutination-inhibiting (HAI) antibody titer rises to A/England/42/72 occurred in 39% of the monovalent Alice strain vaccines; in contrast 18% of those given 2 doses of bivalent Alice/R-75 vaccine and 11% of those given 1 dose of bivalent vaccine had similar four-fold HAI antibody titer rises. HAI antibody titer rises to influenza B/Hong Kong/72 occurred in 38% of R-75 strain monovalent vaccinees, 14% of Alice/R-75 2-dose vaccinees and 11% of Alice/R-75 one dose vaccinees. An epidemic of influenza at onset of the study made evaluation of the efficacy of the vaccine impossible.
Previous work has shown that the sequence leading to exocytosis of the sperm acrosome involves at least three Ca(2+)-requiring processes, the first one probably represented by breakdown of the polyphosphoinositides and the final one by membrane fusion. We have investigated whether phospholipase A2 (PLA2) represents the intermediate Ca(2+)-requiring event by stimulating ram spermatozoa with the ionophore A23187 and various bivalent cations. Spermatozoa prelabelled with [14C]arachidonic acid and treated with ionophore and millimolar Ca2+ showed a considerable release of arachidonic acid; parallel sperm samples similarly treated underwent acrosomal exocytosis. Mn2+ was capable of completely substituting for Ca2+, even if residual Ca2+ in the system was chelated with EGTA: both arachidonic acid release and acrosomal exocytosis took place after treatment with A23187, EGTA and Mn2+. Neither Mg2+ nor Ba2+ promoted arachidonic acid release or exocytosis. The effects of Sr2+ were more complex and allowed us to probe the sequence of events leading to membrane fusion. Both arachidonic acid release and exocytosis occurred after treatment with A23187 and Sr2+ but none of these responses were seen if EGTA was also included. These results suggest that residual micromolar Ca2+ is either needed for Sr2+ to fully promote PLA2 activity, or that micromolar Ca2+ is needed for one or more upstream events that may in turn serve to activate PLA2. Evidence for or against the first possibility was sought by examining PLA2 activity in sperm sonicates. Enzyme activity was maximal in the presence of any bivalent cation and it was not reduced (in the case of Sr2+) or only reduced slightly (Mg2+, Mn2+, Ba2+) if residual Ca2+ was chelated with EGTA; this indicates that Sr2+ can promote PLA2 activity in the total absence of Ca2+. The second possibility was explored by treating spermatozoa with A23187 for 5 min (to allow for complete phosphoinositide breakdown; Roldan and Harrison (1989) Biochem. J. 259, 397-406), and then adding EGTA and Sr2+. This resulted in neither arachidonic acid release nor exocytosis, thus indicating that another as yet unidentified Ca(2+)-dependent event may occur before PLA2 activation.
Ligand binding studies have demonstrated that bivalent cations can alter interactions between purinergic agonists and adenosine receptors in brain membranes. In the present study, we have investigated whether a similar interaction can be demonstrated in terms of a functional response to adenosine, which is the inhibition of synaptic transmission in the CA1 region of the rat hippocampus mediated via presynaptic A1 receptors. Our data suggest that alterations in cation concentrations do not significantly affect the sensitivity of these adenosine receptors, as long as changes are made in such a way as to leave presynaptic Ca2+ entry unaffected. The experimental results do not support the conclusion that there is a specific effect of either Ca2+ or Mg2+ on adenosine receptor sensitivity, such as has been described for agonist interactions with adenosine A1 receptors. We conclude that the effects of bivalent cations observed in ligand binding studies probably reflect an effect at an intracellular site, either on the receptor itself, or perhaps on associated GTP binding proteins.
We cytologically characterized the nucleolar organizer region (NOR) on the bivalent in the yeast Saccharomyces cerevisiae. We used staining with 4'-6-diamidino-2-phenylindole (DAPI), chromomycin A3, and silver nitrate and in situ hybridization technique and utilized a video-intensified microscope system with an ultra-high-sensitive video camera. The results showed that of 16 bivalents of S. cerevisiae, the longest was a recognizable nucleolar chromosome which has an annular and synaptonemal complexless NOR in its submedian portion. The NOR was comprised of 2.65 X 10(9) D DNA which corresponded to 118 copies per haploid of rDNA repeating units. This evidence is discussed in terms of the possible participation of the annular NOR in suppressing the meiotic recombination of the rDNA gene clusters.
The phase behavior of isolated photoreceptor membrane lipids is further investigated by 31P-NMR, in view of earlier discrepant results [(1979) Biochim. Biophys. Acta 558, 330-337; (1982) FEBS Lett. 124, 93-99]. We present evidence that the discrepancy is due to bivalent cations. When resuspended in aqueous media at neutral pH in the absence of bivalent cations, the isolated photoreceptor membrane lipids largely adopt the bilayer configuration. However, upon addition of such cations (Ca2+ greater than Mg2+) or when resuspended in their presence, the formation of other phases (hexagonal H11, lipidic particles) results. The rate of this transition depends on cation concentration and temperature. The transition is not easily reversed by addition of EDTA. Implications with regard to photoreceptor membrane structure and function need further study.
The toxicity of chromium and tin on growth, uptake of NO3- and NH4+, nitrate reductase and glutamine synthetase activity of Anabaena doliolum, and its interaction with bivalent cations, viz. Ca2+, Mg2+, Mn2+, Ni2+, Co2+, and Zn2+, has been studied. Some interacting cations, viz. Ca, Mg, and Mn, substantially antagonized the toxic effects of chromium and tin with reference to growth and nutrient (NO3- and NH4+) uptake in the hierarchical sequence Ca greater than Mg greater than Mn, whereas the sequence of hierarchy was Mn greater than Mg greater than Ca for nitrate reductase and glutamine synthetase activity of A. doliolum. A synergistically inhibitory pattern of interaction was noted for all the parameters, viz. growth, uptake of NO3- and NH4+, nitrate reductase and glutamine synthetase activity of A. doliolum, when Ni, Co, and Zn were used in combination with test metals in the growth medium. These bivalent cations followed the synergistic inhibition sequence Ni greater than Co greater than Zn and potentiated the toxicity of test metals in the N2-fixing cyanobacterium under study.
We have developed a method for use in investigating factors controlling the binding and cross-linking by bivalent haptens of immunoglobulin E (IgE) bound to receptors on rat basophilic leukemia (RBL) cells. This method employs monoclonal anti-2,4-dinitrophenyl (DNP) IgE that is labeled with fluorescein-5-isothiocyanate (FITC), and it measures FITC quenching that accompanies DNP occupation of the antibody combining sites in a titration experiment. The validity of this approach is demonstrated using the monovalent hapten DNP-L-lysine. The affinity constant for this ligand obtained by the FITC quenching method is compared with those obtained with previously established methods: equilibrium dialysis and quenching of endogenous tryptophan for IgE in solution and [3H]-DNP-L-lysine binding to IgE on cells. The FITC quenching method has been used to carry out a detailed study of the binding of monovalent DNP-aminocapryol-L-tyrosine (DCT) and bivalent (DCT)2-cystine to FITC-IgE and its Fab fragments in solution. Intrinsic (K) and cross-linking (Kx) affinity constants are obtained by analyzing the binding curves in terms of simple equilibrium equations. With these DCT haptens the ability of this method to assess hapten binding and cross-linking of IgE bound to receptors on RBL cells is shown.
Although it is recognized that the bivalent binding of an antibody to a multivalent antigen cannot be characterized by true affinity, "functional affinity" is frequently determined. "Functional affinity" is calculated by the same methods used for true affinity, and is presumed to have similar biological significance. The data presented herein demonstrate, for IgG antibodies binding to cell surface antigens, that "functional affinity" does not adequately describe the interactions that occur. First, the dissociation rate was not first-order, but rather reflected heterogeneity of bound Ab. A minority of bound Ab (probably monovalently bound) dissociated relatively rapidly, while the majority dissociated very slowly, and, for most purposes, should be considered irreversibly bound. Although irreversible binding is incompatible with a state of equilibrium, the dissociation rate was greatly increased in the presence of free Ab, which may explain why equilibrium is reached in a Scatchard-type experiment. In addition, "functional affinity" was found to vary depending on trivial experimental conditions, such as the volume of incubation. Thus, antibodies do not possess a single "functional affinity", but rather a spectrum of "functional affinities", which are therefore of little value in comparing different antibodies and cannot be used to predict the extent of binding under other experimental conditions. Both of these discrepancies can be attributed to the effect of bivalent Ab binding. We conclude that "functional affinity" determination is inappropriate.
Hirudin derivatives (e.g. lepirudin, desirudin) and hirudin analogues (e.g. bivalirudin) are bivalent direct thrombin inhibitors; that is, they bind to two distinct sites on thrombin-its active (catalytic) site and its fibrinogen-binding site (exosite 1). These bivalent binding properties contribute to their high affinity and high specificity for thrombin. This review compares the pharmacological properties of these agents, and describes studies of their efficacy and safety in diverse clinical settings such as immune heparin-induced thrombocytopenia, postoperative antithrombotic prophylaxis, and treatment of acute coronary syndrome. Certain disadvantages of hirudin, such as its predominant renal excretion and immunogenicity, have been overcome through development of the hirudin analogue, bivalirudin. Compared with hirudin derivatives, bivalirudin exhibits a shorter half-life (25 vs 80 minutes), predominant non-renal (enzymic) metabolism, and low immunogenicity. Further work is required to define the scope of clinical thrombosis problems that could benefit from these novel agents.
A series of bivalent ligands 6a-d of epibatidine were synthesized. All four ligands showed nanomolar binding affinities at six neuronal nicotinic acetylcholine receptor (nAChR) subtypes in competition binding assays. In contrast to epibatidine, these bivalent ligands are weak partial agonists at the alpha3beta4 nAChR as shown by functional assays.
The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands 2 and 3 were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity as illustrated for alpha5 subtype selective ligand 2 (15 nM). Bivalent ligand 3 with the 5 atom linker folded back upon itself both in solution and in the solid state, moreover, it did not bind to Bz receptors.
The concept of bivalent polypeptides with controllable flexible linkers is demonstrated through the design of a new generation of 'antidote'-reversible inhibitors of thrombin. These molecules contain two binding moieties, each of which in isolation has only a moderate affinity of binding, which are linked together by a flexible peptide bridge. We show that activities of the potent bivalent inhibitors of thrombin can be reversed by the specific, but much weaker, binding of the linker moiety to protein 'antidotes'.
OBJECTIVE: To find the most informative method of XY bivalent detection for spermatozoa presence in testicular tissue of nonobstructive azoospermic men. DESIGN: Prospective study. SETTING: Institute for the Study of Fertility, affiliated with a university medical faculty. PATIENT(S): Thirty-five men with azoospermia, divided into subgroups: complete maturation arrest (n = 10), mixed atrophy (n = 14), and obstructive azoospermia (n = 11). INTERVENTION(S): Testicular tissue biopsies for sperm extraction. MAIN OUTCOME MEASURE(S): Histopathologic and cytology analyses and the presence of XY bivalent formation by fluorescence in situ hybridization probes for centromere and subtelomere regions. Immunostaining of gamma-H2AX for sex body (SB) identification was also performed. RESULT(S): Percentage of spermatocytes with X-Y pairing, determined by the paired short arms pseudoautosomal region, was significantly higher than percentage of spermatocytes with long arm telomeres in proximity in all three groups. The parameter of q telomeres in proximity was the most sensitive index to distinguish one group from the other. Stained SB by gamma-H2AX was found to be the most informative for the prediction of successful sperm retrieval. CONCLUSION(S): Alignment of the X and Y axes that occurs in the late zygotene stage probably precedes the stage in which the SB is stained by gamma-H2AX. Consequently, because of the nonhomogeneity of the testis, when histology raises suspicion of complete maturation arrest percentage of spermatocytes with stained SB is the most informative parameter for sperm presence on sperm retrieval.
Phage display techniques identified a scFv, 15-9, which binds to murine laminin-1 and accumulated selectively in tumors. In this study, a covalent diabody was constructed by changing the amino acid residues at positions VH44 and VL100 to cysteine residues so that the diabody form could be stabilized via a disulfide bond. The covalent diabody was expressed in Pichia pastoris and purified by affinity chromatography. The binding properties were measured by surface plasmon resonance and solid phase binding of (125)I diabody and scFv. Data from the plasmon resonance method yielded calculated K(D)s of 4.4 x 10(-10) M for the covalent diabody and 9.9 x 10(-8) M for the scFv. K(D)s calculated from solid phase binding of radioiodinated proteins were 1.7-2.1 x 10(-10) M and 2.1-2.4 x 10(-8) M respectively. The rate of dissociation of (125)I scFv from solid phase laminin was independent of laminin concentration; however, the dissociation of the (125)I diabody was dependent both on the concentration of laminin and on the concentration of the diabody. Specifically, high concentrations of laminin yielded very slow rates of diabody dissociation indicating that bivalent attachments had formed. When higher amounts of diabody were used that essentially saturated the laminin sites with univalent binding, the dissociation rate was similar to that for the scFv indicating univalent binding. Biodistribution studies in tumor-bearing SCID mice showed that the covalent diabody improved the ratio of tumor/muscle 2 fold over that obtained with the scFv, although the absolute amount of protein bound to the tumor site was not significantly different for the two forms. The data also showed that retention of the diabody in the tumor and kidney, sites where laminin is present in high concentration, was much longer compared to that of scFv. These data are consistent with the hypothesis that both scFv and diabody forms bind to available laminin in vivo with similar association kinetics, but that in situations of high target concentration, the diabody can bind bivalently and is thus retained at the binding site much longer than the scFv.