Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.     

Diversity in the immune system: “Preconceived ideas” or ideas preconceived?

Two concepts of the evolution and regulation of expression of the combining site repertoire of the immune system, are compared. One view is based on the Associative Recognition Theory as formulated by the author and the other is based on the Idiotype Network Idea as conceived by Jerne. The two concepts are analyzed from the point of view of their logic, internal consistency and factual support.     

氨基酰化酶中金属锌离子的功能作用

 氨基酰化酶是含锌金属酶。该酶每摩尔蛋白中含2摩尔Zn(Ⅱ)离子。金属鳌合剂与酶作用,通过竞争螯合Zn(Ⅱ)离子使酶活力下降。残余活力与残留金属含量呈正相关。竞争螯合的结果,生成不含金属的脱辅基酶蛋白,并导致酶活力的丧失。脱辅基酶由于加入Zn(Ⅱ)离子而恢复其活力。实验表明金属锌离子是氨基酰化酶催化活力所必需。与Zn(Ⅱ)离子相似,Co(Ⅱ)离子也可与脱辅基酶相结合并使之复活。 在190—240nm区域内对比了天然酶、脱辅基酶蛋白与Co(Ⅱ)置换氨基酰化酶的圆二色谱。远紫外圆二色谱表明,与天然酶相比,在脱辅基酶中由于金属离子的丧失导致主链构象发生变化,其中α螺旋增加约7％。因而锌离子(钴离子)对蛋白主链的反应最适构象有一定的稳定作用。脱辅基酶与Co(Ⅱ)离子结合,酶的主链构象恢复至与天然酶几近相同。可认为这是促使酶复活的内在因素。     

Lignans of Piper clusii

From the petrol extract of Piper clusii five lignans were isolated. One of the lignans (?)-clusin is assigned the structure (?)-2-furanol-4(1,3-benzodioxol-5-ylmethyl) tetrahydro-3(3,4,5-trimethoxyphenyl) methyl. This is the first report of this compound from a natural source. Asaronaldehyde and sitosterol were also present.     

A note on sequence-dependence of DNA structure

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.     

Lymphocyte subpopulations of regional lymph nodes in human colon and gastric adenocarcinomas

 In order to study the host immune response to tumours, previous knowledge of the cellular composition of regional draining lymph nodes is necessary. Enlarged regional lymph nodes are a common finding in colon and gastric adenocarcinomas. We have studied the cellular composition of normal non-reactive and of regional draining lymph nodes of colon and gastric adenocarcinomas. In normal non-reactive lymph nodes, T lymphocytes (CD2⁺, CD7⁺) constituted the largest fraction of the lymphoreticular cells. These lymphocytes were mainly CD4⁺, and there were more cells expressing the CD45RA isoform of the CD45 antigen than CD45RO. Reactive lymph nodes presented a decreased proportion of CD4⁺ CD45RA⁺ cells and an increased number of B cells. Although most of the T cells in the reactive nodes were CD4⁺ CD45RO⁺, their proportion was similar to that found in normal non-reactive nodes. We studied the presence of the molecules CD28 and CD80 involved in the processes of interaction and activation of T and B lymphocytes. The CD28 molecule was found in all the T lymphocytes, while the CD80 molecule was weakly expressed on the B lymphocyte membrane. Received: 4 January 1996 / Accepted: 28 May 1996     

Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin-2 (IL-2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.     

Constitutively hyposialylated human T-lymphocyte clones in the Tn-syndrome: binding characteristics of plant and animal lectins

Previously, 1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnheret al. (1992)Eur J Immunol 22: 1835–42], Tn+ T lymphocytes express only Tn antigen (GalNAc1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Ac2,6GalNAc1-O-R) or TF (Gal1-3GalNAc1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins:Amaranthus caudatus (ACA),Maackia amurensis (MAA),Limax flavus (LFA),Sambucus nigra (SNA) andTriticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that thein vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol