Double Rosette Research Articles

Double rosette cataract: a case report

Cataract is commonly seen in the elderly population and is a major cause of blindness in Malaysia. The most common morphological types of cataracts associated with the elderly are cortical, nuclear, and posterior subcapsular cataract. Rosette cataracts are commonly associated with blunt trauma. We report a case of a patient who presented with unilateral, non-traumatic, double rosette cataract. He successfully underwent cataract extraction with posterior chamber intraocular lens implantation and the final visual outcome was good.

Hydrogen Bonded non-covalent synthesis by reaction of porphyrin appended calix[4]arene with 5,5-diethylbarbituric acid in solution

Calix[4]arene, diametrically substituted at the upper rim, with two porphyrin appended melamine units spontaneously form well-defined double rosette in the presence of 5,5-diethylbarbituric acid. This assembly consists of nine components which utilize 36 hydrogen bonds and are quite stable in apolar solvents of up to 10−4 M concentration. The formation of assembly between porphyrin appended calix[4]arene and diethylbarbiturate has been broadly studied by UV–visible, fluorescence and 1H NMR spectroscopic techniques. The stoichiometries of proposed aggregates have been analyzed by facile titration of calixarene and barbital. Furthermore, the MALDI-TOF mass measurement is found fully compatible with the observed stoichiometries.

Silver Containing Nanostructures from Hydrogen-bonded Supramolecular Scaffolds

The self-organisation of silver-containing hydrogen-bonded rosette assemblies on highly oriented pyrolytic graphite (HOPG) surfaces is described. The introduction of silver atoms into the double rosette architecture was achieved using the affinity of silver cations for cooperative π-donors or cyano functionalities on the double rosettes. Highly ordered 2-D nanorod domains with an inter-row spacing of 4–5 nm oriented in different directions were revealed by tapping-mode atomic force microscopy (AFM). This new and simple strategy for the creation of metal-containing supramolecular nanorod arrays that can act as well-defined surface-immobilised self-assembled scaffolds, will contribute to the development of functionalised nanoarchitectures via bottom–up approaches.

Gold Nanoparticle Assemblies through Hydrogen-Bonded Supramolecular Mediators

The synthesis of spherical gold nanoparticle assemblies with multicomponent double rosette molecular boxes as mediators is presented. These nine-component hydrogen-bonded supramolecular structures held together by 36 hydrogen bonds induce gold nanoparticle assembly. The morphologies of the nanoparticle assemblies can be tuned easily by changing the quantity of the building block chemisorbed on the nanoparticle surface.

Co-operative Stabilisation of a 15-component Nanostructure

2-Amino-4-(4- tert-butylphenylamino)-6-{4-[(4-pyridyl)ethynyl]phenylamino}-1,3,5-triazine was synthesised and mixed with dibutylbarbituric acid in CDCl3 to study rosette formation. The formation of a double decker rosette aggregate was studied by complexation of the pyridyl groups to a zinc containing porphyrin dimer.

Amplification of Chirality in Hydrogen‐Bonded Tetrarosette Helices

The amplification of chirality in hydrogen-bonded tetrarosette assemblies under thermodynamic equilibrium is described. The extent of the chiral amplification obtained by means of "sergeants-and-soldiers" experiments depends only on the structure of the assembly and it is independent of the methodology used for the formation of the tetrarosette assemblies. The difference in free energy (deltaG(o)(M/P)) between the M- and P-diastereomeric helices is up to 40 times higher than for double rosette assemblies.

Self-assembly and stability of double rosette nanostructures with biological functionalities

The syntheses of calix[4]arene dimelamines that are functionalized with alkyl, aminoalkyl, ureido, pyridyl, carbohydrate, amino acid and peptide functionalities, and their self-assembly with barbituric acid or cyanuric acid derivatives into well-defined hydrogen-bonded nanostructures are described. The thermodynamic stability of these hydrogen-bonded assemblies was studied by CD spectroscopy in mixtures of CHCl3 and MeOH. The stability of the assemblies depends on several steric factors and the polarity of the functional groups connected to the assembly components.

Covalent Linkage of Melamine and Cyanurate Improves the Thermodynamic Stability of Hydrogen‐Bonded Double Rosettes in Polar Solvents

AbstractThis paper describes studies on the synthesis, self‐assembly behavior, and thermodynamic stabilities of eight different calix[4]arene di(melamine‐cyanurate) or di(melamine‐barbiturate) conjugates. The successful synthetic strategy comprises the preparation of amino‐n‐alkyl‐functionalized calix[4]arene dimelamines coupled with a carboxyl‐functionalized cyanurate or barbiturate by an amide bond‐forming reaction. 1H NMR experiments show that three of the eight conjugates form well‐defined double rosette assemblies. DMSO titration experiments illustrate that the covalent linkage between the cyanurate and dimelamine moieties produces a significant increase in the thermodynamic stabilities of these conjugates. The relative stability of the assemblies seems to be primarily governed by the structure of the component connecting the melamine and cyanurate units. The highest stability (χDMSO = 70%) was observed for the di(melamine‐cyanurate) assembly in which the components are connected through an n‐heptylamidomethyl linker. Increasing the linker size from n‐heptylamidomethyl to n‐decylamidomethyl slightly reduced the thermodynamic stability (ΔχDMSO ≈︁ 10%) of the corresponding double rosette assembly. The thermodynamic stability of the double rosette structure decreases drastically, however, on introduction of substituents at the position α to the methylcarbonyl group. With benzyl substituents (based on D‐phenylalanine), neither the n‐heptyl‐ nor the n‐decylamidomethyl‐linked compounds any longer form the double rosette structure in solution. For the conjugates with a methyl substituent at this position (based on L‐alanine), only the heptyl‐linked compound still forms the double rosette structure, the χDMSO value being reduced in this case by 15%. Neither of the di(melamine‐barbiturate) conjugates form double rosette structures at all. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Growth of individual hydrogen-bonded nanostructures on gold monolayers.

The growth of individual nanometer-sized (3.4 +/- 1.4 nm) hydrogen bonded assemblies 1(2) x (DEB)6 on gold monolayers was achieved through an exchange reaction between single isolated calix[4]arene dimelamine 2 (1.1 +/- 0.2 nm) embedded in hexanethiol monolayers and double rosette hydrogen bonded assembly 1(3) x (DEB)6 in solution. The growth process was monitored by tapping mode atomic force microscopy (TM-AFM).

Complexation of phenolic guests by endo- and exo-hydrogen-bonded receptors.

This article describes the complexation of phenol derivatives by hydrogen-bonded receptors. These phenol receptors are formed by self-assembly of calix[4]arene dimelamine or tetramelamine derivatives with 5,5-diethylbarbiturate (DEB) or cyanurate derivatives (CYA). The double rosette assemblies 3(3).(DEB)6/(CYA)6 have their phenol-binding functionalities (ureido groups) at the top and at the bottom of the double rosette (exo-receptors). The tetrarosette assemblies 4(3).(DEB)12/(CYA)12 form a cavity with binding sites between the two double rosettes for guest encapsulation (endo-receptors). An intrinsic binding constant Ka of 202 M-1 and 286 M-1 for the binding of 4-nitrophenol to the ureido functionalized exo- and endo-receptors, respectively, was observed. For the exo-receptor a 1:6 stoichiometry was observed while for the endo-receptor 1:4 binding stoichiometry was determined by Job plot and MALDI-TOF MS. The important role that the hydroxy group's acidity plays in the complexation of 4-nitrophenol is clarified by binding studies with different phenol derivatives. The hydrogen-bonded receptors showed a much smaller response towards less acidic phenol derivatives.

Enantioselective formation of a dynamic hydrogen-bonded assembly based on the chiral memory concept.

In this paper, we report the enantioselective formation of a dynamic noncovalent double rosette assembly 1a(3).(CYA)(6) composed of three 2-pyridylcalix[4]arene dimelamines (1a) and six butylcyanuric acid molecules (BuCYA). The six 2-pyridyl functionalities of the assembly interact stereoselectively with chiral dicarboxylic acids 3a-e via two-point hydrogen-bonding interactions. One of the two enantiomeric assemblies (P- or M-) 1a(3).(CYA)(6) is formed in excess as the result of the complexation of the chiral diacids, resulting in formation of optically active assemblies. The complexations with dibenzoly tartaric acids D-3a and L-3a (3 equivalent), respectively, leading to the formation of diastereomeric assemblies (P)-1a(3).(BuCYA)(6).(D-3a)(3) and (M)-1a(3).(BuCYA)(6).(L-3a)(3) with 90% diastereomeric excess. The diastereomeric excess in (M)-1a(3).(BuCYA)(6).(L-3a)(3) is "memorized" when L-3a is removed by precipitation with ethlylenediamine (EDA). The assembly (M)-1a(3).(BuCYA)(6) is still optically active (90% enantiomeric excess), although none of its individual components are chiral. (M)-1a(3).(BuCYA)(6) has a high kinetic stability toward racemization (E(a) = 119 kJ mol(-)(1), half-life of (M)-1a(3).(BuCYA)(6) is ca. 1 week at 20 degrees C).

Interaction between rat peritoneal macrophages and sensitised erythrocytes: Dependence on IgG subclass, antibody density and the degree of hapten conjugation

The interaction between macrophages (MØ) and antibody sensitised target cells was studied by the use of rat peritoneal macrophages, TNP hapten conjugated sheep red blood cells (SRBC) and homologous antibodies of subclasses IgG1 and IgG2a. Under optimal conditions, the great majority of the MØ formed rosettes with IgG1-sensitised antibodies while a maximum of 50% was achieved when target cells were sensitised by IgG2a. Using a double rosette technique, the major part of rosette-forming cells was found to bind both of the isotypes. IgG1-mediated rosette formation was observed at very low degrees of sensitisation as opposed to IgG2a-mediated target cell binding. Not only the amount of bound antibody but also the degree of hapten conjugation (epitope density) appear to influence the ratio of rosette-forming cells. IgG1-mediated rosette formation was partially inhibited by monomeric IgG1 and more efficiently by soluble ovalbumin (OVA)-anti-OVA complexes involving IgG1-type antibodies, while IgG2a mediated rosette formation was inhibited by OVA-anti-OVA complexes containing IgG2a type antibodies, and less efficiently by complexes involving IgG1. No inhibition was found by monomeric IgG2a. Based on the present data, we propose that two types of receptors are involved in the interaction of MØ and target cells coated by IgG1 and/or IgG2a type antibodies. One requires a multiple antibody-receptor interaction, binding both subclasses at overlapping binding sites; the other is able to interact with IgG1 and does not depend on the multiplicity of interactions.

Vaccination with Mycobacterium bovis BCG affects the distribution of Fc receptor-bearing T lymphocytes in experimental pulmonary tuberculosis

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated and challenged 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using a double rosette assay with isotype-specific antibody-coated ox and uncoated rabbit erythrocytes, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (IgG) (T gamma cells) or IgM (T mu cells) were quantified in tissues taken from animals that were killed within 4 weeks postchallenge. Tuberculin reactivity in vivo and in vitro and antimycobacterial resistance were also measured. BCG vaccination protected the guinea pigs and resulted in significantly enhanced proportions of T mu cells in the blood during the first 3 weeks and in the spleen during weeks 2 and 3 postchallenge. Levels of T gamma cells declined in all tissues during the first 3 weeks of infection and were unaffected by prior vaccination with BCG. Increased proportions of T mu cells in the blood were accompanied by dramatic tuberculin skin reactions and purified protein derivative-induced lymphoproliferation in BCG-vaccinated guinea pigs during the first 2 weeks following virulent pulmonary challenge. Peak levels of T mu cells in the spleens of vaccinated animals at 2 weeks coincided with the first appearance of virulent mycobacteria in that organ. BCG vaccination appears to influence immunoregulatory events in pulmonary tuberculosis through effects on the distribution of IgM Fc receptor-bearing (T mu cell) T lymphocytes.

膀胱癌患者におけるIgG-Fcレセプター陽性T細胞の動向

IgG-FcR+-T cell ratio was measured in 50 patients with bladder tumor as a parameter to estimate the cell mediated immunity before the treatment. IgG-FcR+-T cell was detected by the double rosette method, employing sheep erythrocytes and rabbit IgG antibody coated chicken erythrocytes. IgG-FcR+-T cell ratio in 50 patients with bladder tumor was 7.6 +/- 5.7% (mean +/- standard deviation), while that in controls consisting of 20 patients with other diseases but bladder tumor was 5.2 +/- 2.4%. There was no significant difference between them. IgG-FcR+-T cell ratio in the group in early stages (pTa, pT1 & pT2) was 5.4 +/- 4.5%, and that in the group in advanced stages (pT3a, pT3b & pT4) was 10.8 +/- 6.0%.--significant difference between them (p less than 0.001)--. IgG-FcR+-T cell ratio in the low graded group (G0 & G1) was 5.5 +/- 4.4% and that in the high graded group (G2 & G3) was 9.7 +/- 6.2%.--significant difference between them (p less than 0.01)--. IgG-FcR+-T cell ratio in patients with the serum level of CEA over 2.6 ng/ml was 12.5 +/- 6.3% and that under 2.5 ng/ml was 5.6 +/- 3.7%.--significant difference between them (p less than 0.001)--. In patients having IgG-FcR+-T cell ratio over 9%, there was a correlation between IgG-FcR+-T cell ratio and PHA-induced lymphocyte blastogenesis ratio (r = -0.81, p less than 0.01). In patients with the serum level of CEA over 2.6% ng/ml, PHA-induced lymphocyte blastogenesis was decreased significantly and IgG-FcR+-T cell ratio was increased significantly. These findings suggest that serum CEA may play a role to proliferate the IgG-FcR+-T cells in association with an inhibition of PHA-induced lymphocyte blastogenesis.

Sequential changes of the T Lymphocytes followingE R Rosette formation in guinea pigs. I. Expression of Fc and Complement Receptors

Changes in proportions of the Fc and complement receptor (FcR, CR) positive T lymphocytesfrom guinea pigs following their interaction with rabbit erythrocytes (ER) were studied using EA and EAC rosette forming assays. Significant increases in the percentages of EA and EAC rosette forming cell (RFC) were observed when thymocytes or lymph node cells were assayed after ER rosette formation. Furthermore, T-enriched fraction by the E R monolayer adherence technique also showed similar or somewhat higher increases in the proportions of both EA and EAC RFC than those of unfractionated cells after contact with ER. The double rosette assay by ER with EA or EAC showed that 50–80 % of the Fc and/or complement receptor positive lymphocytes bound rabbit erythrocytes simultaneously. These findings strongly suggest that at least a subset of the guinea pig T cells is altered to express Fc and/or complement receptors on their surfaces following the interaction with E R.

T Cell Subset and Steroid Sensitivity in Idiopathic Nephrotic Syndrome in Children

AbstractThe relationship between T cell subset of peripheral blood lymphocytes and steroid sensitivity was studied in children with idiopathic nephrotic syndrome (INS). The subjects were 28 INS children, aged 2 to 16 years. T cells bearing receptors for IgG (Tγ) were identified by double rosette assay with sheep erythrocytes and IgG‐sensitized ox erythrocytes. T cells bearing receptors for IgM (Tμ) were identified by IgM‐sensitized ox erythrocytes rosette assay in purified T cells after incubating overnight.In steroid sensitive INS children, decreased numbers of Tγ cells and increased numbers of Tμ cells in onset or relapse were found and they were normalized with corticosteroid administration. Tγ cells of steroid nonsensitive INS children remained decreased even after steroid therapy.Tγ cells have been suggested to be, at least in part, suppressor T cells. Alteration of peripheral blood Tμ cells in this observation therefore may suggest the abnormality of suppressor T cells in INS children.

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells.

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.

Detection of guinea pig Tμ and Tγ cells by a double rosette assay

By a rosette assay with ox red blood cells (ORBC) sensitised with rabbit IgM and IgG antibodies (EAμ and EAγ), receptors for IgM (Fcμ-R) and IgG (Fcγ-R) on the surface of guinea pig lymphocytes in the different lymphoid organs were demonstrated. A double rosette assay with rabbit erythrocytes (RRBC) and EAμ or EAγ for detection of T cells with Fcμ-R (Tμ) or Fcγ-R (Tγ) was also developed. Fcμ-R was detected on 40.1%, 48.7%, 35.0% and less than 1% of peripheral blood, lymph node, spleen and thymus cells respectively. By a double rosette assay with RRBC and EAμ, approximately 30% of peripheral blood, lymph node and spleen T cells were identified as Tμ cells. Tγ cells as revealed by a double rosette of RRBC and EAγ were found to be 3% and 5% respectively of peripheral blood and lymph node T cells, whereas much higher numbers of T cells in the spleen (32.7) were identified as Tγ cells. This double rosette assay of Tμ and Tγ cells in the peripheral blood of guinea pigs which had received prednisolone showed that the proportion of Tμ cells decreased early after drug administration and that Tγ cells increased in number. Thus the methods provide a useful means of detecting Tμ and Tγ cells in guinea pig. This assay system for Tμ and Tγ cells in guinea pigs should be useful for studies of their variation or functional significance in the immune response.

膀帯血リンパ球Subpopulation及び男児臍帯血中の母体リンパ球に関する研究

At first, lymphocyte subpopulations were examined in the cord blood by using various technics, which were the E-rosette forming cell, EN-rosette forming cell, early-EN-rosette forming cell, EAC-rosette forming cell, IgG-Fc-receptor binding T cell (double rosette forming cell), and immunofluorescent staining assays, and the results were compared with t hose of adults.In the second study, the possibility of maternofetal transfer of lymphocytes was investigated in the cord blood. Cord lymphocytes were obtained from 15 healthy male newborns and cultured for chromosome analysis. The culture was carried out in many combinations of pH, mitogens, cell numbers and culture time as follows: (1)pH of culture media were 7.2 and 6.8, (2) Mitogens were phytohemagglutinin (PHA), pokeweed mitogen (PWM), and concan avalin A (Con-A),(3) Added cell concentrations (per 5 ml of culture media) were 3×106,2.5×106,5×105, and 1 x 105, (4) Culture time were 72 hours and 96 hours.These four groups of conditions were combined each other and all cultures were done at 37°C in environment of 5 % CO2. Then the cultured cells were prepared for chromosome analysis and karyotyped. (One of them were performed whole blood cultures. )The following results were obtaind.1) In the cord blood, E-, EN-, and early-EN-rosette forming cells (T lymphocytes) decreased in percentage as compared with those of adults.2) The absolute numbers of EN-rosett e forming cells increased in the cord blood compared with those of adults.3) In the cord blood, neuraminidase-treated E rosette forming cells (EN- and early-EN-rosette forming cells) increased in percentage and its standard deviation was smaller than that of the results obtained from neuraminidase-untreated E rosette forming cells (E-rosette forming cells)4) In the cord blood, there was no difference in percentage between EN-rosette forming cells and early-EN-rosette forming cells, which suggests that these two populations were the same lymphocyte-subset (of T cells).5) EAC-rosette forming cells sho wed no difference in percentage between the cord lymphocytes and the peripheral blood lymphocytes of adults, but the absolute numbers of EAC-rosette forming cells increased in the cord blood compared to those of adults.6) The percentage of surface immunoglobulin (SmIg) bearin g lymphocytes (B lymphocytes)were higher in the cord blood than those of adults.7) Surface IgD bearing lymphocytes in the cord blood showed an increased percentage as compared with those of adults.8) In the cord blo od, it is likely that “null cells” (lymphocytes which have no surface marker) increased in percentage compared with those of adults.9) IgG-Fc-receptor binding T lymphocytes examined by double rosette formation showed no significant difference in percentage between cord lymphocytes and adults.10) Chromosome analysis were performed on 1320 metaphases in PWM culture,196 metaphases in PHA culture, and 126 metaphases in Con-A culture, altogether 1642 metaphases from 15 healthy male cord blood. Among these experiments, only 1 XX-karyotype was found among the PWM-stimulated metaphases, which was cultured for 96 hours in culture media of pH 7.2. The results obtained so far suggest that maternal lymphocytes do not pass across the placental barrier in normal pregnancy.

Peripheral blood lymphocyte receptors for B-lymphoblastoid cell lines (B-LCL)

Because interactions between B cells and T lymphoyctes are of fundamental importance in the generation of the immune response to most antigens, we attempted to identify the cells capable of binding B- lymphoblastoid cell lines (B-LCL), their tissue distribution, and their presence in other species. Cells bearing a surface receptor for B-LCL were found in human peripheral blood, tonsil, and bone marrow, as well as mouse and rat spleen. Binding cells were phenotypically heterogeneous. The majority are T cells as defined by their ability to bind sheep red blood cells (E-rosettes). However, a subpopulation of non-T-lymphocytes were capable of binding B-LCL. This was demonstrated by depleting T cells with an E-rosette centrifugation technique and then performing a double rosette assay. The wide distribution of T lymphocytes with receptors for B-lymphoblastoid cells within peripheral lymphoid organs and their presence in several species suggest that these surface molecules may represent one of the means by which T cells and B cells interact in the induction of the immune response to T- dependent antigens.