Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase

In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.     

Mitral annular motion as a surrogate for left ventricular function: correlation with brain natriuretic peptide levels.

BACKGROUND: Pulsed-wave (PW) Doppler tissue velocities of the mitral annulus correlate well with Left Ventricular (LV) diastolic(D) and systolic(S) functions. Brain natriuretic peptide (BNP) levels have been shown to be elevated in patients with symptomatic LV dysfunction (Dys) and correlate to the severity of symptoms and prognosis. OBJECTIVES: To validate the accuracy of mitral annular motion (MAM) assessed by Doppler Tissue Imaging (DTI) & M-mode Echocardiography (MME) as a surrogate for determination of LV function in comparison with BNP. METHODS: A series of 133 patients with a variety of cardiac pathologies referred for echocardiography and 20 healthy age & sex matched volunteers as a control group were included the study. Ejection fraction (EF) of LV, Doppler recordings of the mitral inflow, MME and PWDTI data (from each of 4 mitral annular sites, inferior, anterior, septum and lateral) were obtained. Mean peak (S) MAM velocity (Sm), mean annular early (D) velocity (Em) by PWDTI and mean mitral annular plane (S) excursion (MAPSE) by MME were calculated by averaging of values measured at each annular site. BNP levels were measured by a rapid immunoassay and blinded to cardiologist making the assessment of LV function. RESULTS: MAPSE < 12 mm determined by MME has 90% sensitivity, 88% specificity & 89% accuracy for detection of LVEF <50%, while these values were 94%, 93% & 94% respectively for (Sm) < 8 cm/s determined by PWDTI. BNP level>75 pg/ml has 98% sensitivity, 90% specificity & 97% accuracy for detection of LV Dys either (S,D, or both). BNP levels were significantly higher in patients with combined (S & D) Dys. Than those with only (S) Dys, the later group had significantly higher BNP levels than those with only (D) Dys. (1054.5 +/- 202.3 pg/ml vs. 500 +/- 39.9 pg/ml & 500 +/- 39.9 pg/ml vs. 215.3 +/- 100.9 pg/ml respectively, P < 0.001) & each were significantly higher than control group (12.3 +/- 5.7 pg/ml, P < 0.001). Significant correlations (P < 0.001 for all) were found between BNP levels and Em (r =-0.82), Sm (r=-0.7), early transmitral (E) to Em ratio (r=0.61), MAPSE (r=-0.54), LVEF(r=-0.64) & LV end D dimension (r=0.63). CONCLUSION: MME and PWDTI used for assessment of MAM are useful methods for evaluation of LV function but parameters measured by PWDTI correlate more strongly with plasma BNP levels than those measured by MME and provide a simple, sensitive, accurate and reproducible tool for early diagnosis of LV dysfunction.     

蝎毒和蝎毒多肽对家兔血小板聚集的影响

目的　检测蝎毒 (SV)和蝎毒多肽 (PSV)对家兔凝血系统的影响 .方法　麻醉家兔 ,颈动脉取血制备富含血小板血浆 (PRP) ,分别加入SV和PSV ,测定二磷酸腺苷 (ADP)诱导的血小板聚集作用 .结果　较低浓度的SV即可使血小板聚集曲线明显下降 ;PSV对血小板聚集影响不大 ,即使用较高的浓度 ,血小板聚集曲线亦仅轻微下降 .结论　SV对血小板聚集起抑制作用 (抗凝血作用 ) ;PSV对凝血系统的影响远远小于蝎毒     

Experimental immunotherapeutic approaches for atherosclerosis

Therapeutic options in atherosclerosis have largely been limited to the control of risk factors, such as hypercholesterolemia, hypertension, or diabetes. However, atherosclerosis is a chronic inflammatory disease in which dyslipidemia and inflammation are equally involved in disease pathogenesis. Moreover, abundant epidemiological and experimental evidence point to an important modulatory role of innate and adaptive immunity in atherogenesis, providing novel therapeutic targets for this disease. Indeed, there is now accumulating data in animal models demonstrating the potential for immunotherapeutic approaches to treat atherosclerosis. These include both general and antigen-specific ways of modulating immune functions, and they show great promise for the development of alternative and/or adjuvant therapies for atherosclerosis.     

Anti-coreceptor antibodies profoundly affect staining with peptide-MHC class I and class II tetramers

The T cell coreceptors CD8 and CD4 bind to invariable regions of peptide-MHC class I (pMHCI) and class II (pMHCII) molecules, respectively, and facilitate antigen recognition by a number of mechanisms. It is established that some antibodies (Ab) specific for the CD8 molecule, which stabilizes TCR/pMHCI interactions, can alter the binding of pMHCI tetramers to cell surface TCR. In contrast, the extremely weak pMHCII/CD4 interaction does not stabilize TCR/pMHCII interactions or contribute to cognate tetramer binding; consequently, it is assumed that anti-CD4 Ab do not affect pMHCII binding. Here, we used a panel of point-mutated HLA A2 molecules with a range of affinities for CD8 spanning over three orders of magnitude to demonstrate that anti-CD8 Ab-mediated inhibition of pMHCI tetramer binding and cognate T cell activation correlates directly with the strength of the pMHCI/CD8 interaction. Further, some anti-CD4 Ab were found to block pMHCII tetramer binding; these effects were also paralleled in T cell activation assays. In sum, these data challenge the assertion that anti-coreceptor Ab exert their effects on T cell activation and pMHC binding solely by blocking pMHC/coreceptor interactions.     

Epitope-based approaches to a universal influenza vaccine

The development of vaccines has been one of the most important contributions of immunology to public health to date. Although several infectious diseases have all but vanished thanks to effective vaccines, the most common infectious disease, influenza, still represents a major threat to public health. This is more concerning than ever before in light of potentially virulent avian pandemic strains which have emerged in the last decade and infected human hosts, causing high morbidity and mortality. Despite considerable efforts to improve production of influenza vaccines and vaccinate large portions of the population annually, the currently available influenza vaccines are strain-specific and not effective enough. Considering the vulnerability of infants and elderly to seasonal influenza-related complications and the ever present public health threat of a deadly influenza pandemic, there is urgent need for a new kind of influenza vaccine. Ideally, such a vaccine should provide enhanced long term, multi-strain protection without compromising safety and in this way, dramatically improve global protection against seasonal and pandemic influenza viruses. This review highlights one approach to developing a universal influenza vaccine, which is based on highly conserved viral sequences, ‘epitopes’, that specifically activate humoral and/or cellular immune responses. This approach to vaccinology was pioneered by Prof Arnon, who initiated development of an epitope-based universal vaccine called Multimeric-001 (M-001), which has already been validated in clinical trials to induce broad immunity against A and B-Type, seasonal and pandemic strains.     

Effect of peptide secondary structure on adsorption and adsorbed film properties on end-grafted polyethylene oxide layers

Poly-l-lysine (PLL), in α-helix or β-sheet configuration, was used as a model peptide for investigating the effect of secondary structures on adsorption events to poly(ethylene oxide) (PEO) modified surfaces formed using θ solvents. Circular dichroism results showed that the secondary structure of PLL persisted upon adsorption to Au and PEO modified Au surfaces. Quartz crystal microbalance with dissipation (QCM-D) was used to characterize the chemisorbed PEO layer in different solvents (θ and good solvents), as well as the sequential adsorption of PLL in different secondary structures (α-helix or β-sheet). QCM-D results suggest that chemisorption of PEO 750 and 2000 from θ solutions led to brushes 3.8 ± 0.1 and 4.5 ± 0.1 nm thick with layer viscosities of 9.2 ± 0.8 and 4.8 ± 0.5 cP, respectively. The average number of H₂O per ethylene oxides, while in θ solvent, was determined as ∼0.9 and ∼1.2 for the PEO 750 and 2000 layers, respectively. Upon immersion in good solvent (as used for PLL adsorption experiments), the number of H₂O per ethylene oxides increased to ∼1.5 and ∼2.0 for PEO 750 and 2000 films, respectively. PLL adsorbed masses for α-helix and β-sheet on Au sensors was 231 ± 5 and 1087 ± 14 ng cm⁻², with layer viscosities of 2.3 ± 0.1 and 1.2 ± 0.1 cP, respectively; suggesting that the α-helix layer was more rigid, despite a smaller adsorbed mass, than that of β-sheet layers. The PEO 750 layer reduced PLL adsorbed amounts to ∼10 and 12% of that on Au for α-helices and β-sheets respectively. The PLL adsorbed mass to PEO 2000 layers dropped to ∼12% and 4% of that on Au, for α-helix and β-sheet respectively. No significant differences existed for the viscosities of adsorbed α-helix and β-sheet PLL on PEO surfaces. These results provide new insights into the fundamental understanding of the effects of secondary structures of peptides and proteins on their surface adsorption.     

抗肿瘤多肽类药物的来源及其机制的研究进展

恶性肿瘤已成为严重影响人类健康的一种疾病,其治疗手段一直是人们所探索的内容,药物治疗就是其中一种治疗方法。与传统化疗药物相比,抗肿瘤多肽类药物以其分子量小、特异性强、毒性低等特点作为新的治疗肿瘤药物一直广受人们关注。多肽的来源广泛,有存在于天然动植物及微生物体内,也可以通过蛋白质酶解或人工合成得到。近年来,越来越多的多肽类药物被发现除抗菌外还具有抗肿瘤的作用,其抗肿瘤的机制多种多样但尚未完全清楚,是许多研究者研究的重点。本文从多肽类药物的来源与特点及它们的抗肿瘤机制等方面对多肽类药物的研究进展进行综述。     

Induction of specific and flavivirus—Cross-reactive CTLs by immunization with a single dengue virus-derived CTL epitope peptide

Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.