A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified β-cyclodextrin: Insight into its intramolecular interactions

Background

Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose–response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed.

Results

In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified β-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly.

Conclusions

The study of a LHRH analogue conjugated with modified β-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery.

General significance

NMR in combination with MD simulation is of great value for a successful rational design of peptide–cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.     

Interfacial instability and membrane internalization in human erythrocytes heated in the presence of serum albumin

The dynamics of morphological change, when human erythrocytes are heated through the spectrin denaturation temperature in the presence of bovine serum albumin, has been studied using differential interference contrast optics and a television video analysis system. Most washed (control) cells developed a wavy disturbance, with an average of $\text{6.6 ± 0.4}$ (2 S.E.) waves per cell rim, when heated. The average number of waves per cell rim decreased and the percentage of heated cells showing morphological changes in the dimple region increased with increasing serum albumin concentration, reaching 100% at 1.0 g/l. The change in the dimple region of cells heated in the presence of serum albumin involved the growth of a regular wavy disturbance around the cell dimple rim. The development of the wavy disturbance on the dimple, which resulted in the internalization of membrane, has been examined as an example of an interfacial instability on a biological membrane. Scanning and transmission electron micrographs confirm membrane internalization.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Exacerbation of vascular endothelial injury in the generalized Shwartzman reaction by the administration of anti-E-selectin antibody

Previously, we reported that the consecutive administration of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) induced systemic injury of vascular endothelial cells. The aim of this study was to investigate the participation of vascular adhesion molecules in the vascular endothelial injury of GSR. The administration of anti-E-selectin antibody in GSR-induced mice resulted in massive apoptosis of vascular endothelial cells and congestion in blood vessels. Further, marked hemorrhage was found in the pulmonary alveoli of those mice. GSR, especially lung injury, was definitely exacerbated by the administration of anti-E-selectin antibody. On the other hand, the administration of anti-VCAM-1 antibody did not induce such injury of vascular endothelial cells. The possible role of E-selectin in the exacerbation of vascular endothelial injury in GSR is discussed.     

竹叶青蛇咬伤致DIC的救治体会

目的观察中西医结合救治竹叶青蛇咬伤致DIC的疗效。方法采用抗蝮蛇毒血清结合清热解毒、凉血止血、活血化瘀的中药治疗竹叶青蛇咬伤致DIC的患者13例。结果13例患者全部治愈。结论早期应用抗蝮蛇毒血清联合中药治疗竹叶青蛇咬伤致DIC的疗效确切。     

Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

蛇伤凉血合剂对竹叶青蛇咬伤致DIC患者凝血机制的影响

目的观察蛇伤凉血合剂对竹叶青蛇咬伤致DIC患者凝血机制的影响及临床疗效。方法将14例患者随机分为治疗组及对照组,均给予常规治疗,治疗组加服蛇伤凉血合剂180ml,每日2次;于就诊时、给药后72h检测血小板（PLT）、凝血酶原时间（PT）、部分活化凝血活酶时间（APTT）、纤维蛋白原（Fg）、纤维蛋白降解产物（FDP）及血浆鱼精蛋白副凝固试验（3P）、D-二聚体（D-Dimer）变化．同时观察患者出血情况。结果治疗72h后PLT、PT、APTT、Fg、FDP两组比较,差异有显著性或非常显著性意义（P〈0．05）;D-Dimer、3P试验．两组均转为阴性。结论蛇伤凉血合剂对竹叶青蛇咬伤致DIC患者疗效显著,且安全、可靠,对保护正常凝血机制有一定的作用。     

Dissolved carbon leaching from soil is a crucial component of the net ecosystem carbon balance

Estimates of carbon leaching losses from different land use systems are few and their contribution to the net ecosystem carbon balance is uncertain. We investigated leaching of dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and dissolved methane (CH₄), at forests, grasslands, and croplands across Europe. Biogenic contributions to DIC were estimated by means of its δ¹³C signature. Leaching of biogenic DIC was 8.3±4.9 g m^?2 yr^?1 for forests, 24.1±7.2 g m^?2 yr^?1 for grasslands, and 14.6±4.8 g m^?2 yr^?1 for croplands. DOC leaching equalled 3.5±1.3 g m^?2 yr^?1 for forests, 5.3±2.0 g m^?2 yr^?1 for grasslands, and 4.1±1.3 g m^?2 yr^?1 for croplands. The average flux of total biogenic carbon across land use systems was 19.4±4.0 g C m^?2 yr^?1. Production of DOC in topsoils was positively related to their C/N ratio and DOC retention in subsoils was inversely related to the ratio of organic carbon to iron plus aluminium (hydr)oxides. Partial pressures of CO₂ in soil air and soil pH determined DIC concentrations and fluxes, but soil solutions were often supersaturated with DIC relative to soil air CO₂. Leaching losses of biogenic carbon (DOC plus biogenic DIC) from grasslands equalled 5–98% (median: 22%) of net ecosystem exchange (NEE) plus carbon inputs with fertilization minus carbon removal with harvest. Carbon leaching increased the net losses from cropland soils by 24–105% (median: 25%). For the majority of forest sites, leaching hardly affected actual net ecosystem carbon balances because of the small solubility of CO₂ in acidic forest soil solutions and large NEE. Leaching of CH₄ proved to be insignificant compared with other fluxes of carbon. Overall, our results show that leaching losses are particularly important for the carbon balance of agricultural systems.