Interaction between phosphatidylserine vesicles and rat brain synaptosomes

Five different DNA sequences of Phanerochaete chrysosporium capable of supporting autonomous replication of yeast integration plasmid (YIp5) in Saccharomyces cerevisiae were isolated. These hybrid plasmids with the autonomous replication sequences from P. chrysosporium are maintained extra-chromosomally, are mitotically unstable and transform Ura3 deletion mutant of S. cerevisiae to Ura+ phenotype with high frequency. The autonomous replication sequence in pRR2, one of the recombinant plasmids, was further characterized and was shown to be homologous to P. chrysosporium genomic DNA. Restriction analyses showed that this plasmid has unique PvuII and SalI restriction sites for cloning.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Identification of the minimum pharmacophore of lipid–phosphatidylserine (PS) binding peptide–peptoid hybrid PPS1D1

We previously reported a unique peptide–peptoid hybrid, PPS1 that specifically recognizes lipid–phosphatidylserine (PS) and a few other negatively charged phospholipids, but not neutral phospholipids, on the cell membrane. The dimeric version of PPS1, i.e., PPS1D1 triggers strong cancer cell cytotoxicity and has been validated in lung cancer models both in vitro and in vivo. Given that PS and other negatively charged phospholipids are abundant in almost all tumor microenvironments, PPS1D1 is an attractive drug lead that can be developed into a globally applicable anti-cancer agent. Therefore, it is extremely important to identify the minimum pharmacophore of PPS1D1. In this study, we have synthesized alanine/sarcosine derivatives as well as truncated derivatives of PPS1D1. We performed ELISA-like competitive binding assay to evaluate the PS-recognition potential and standard MTS cell viability assay on HCC4017 lung cancer cells to validate the cell cytotoxicity effects of these derivatives. Our studies indicate that positively charged residues at the second and third positions, as well as four hydrophobic residues at the fifth through eighth positions, are imperative for the binding and activity of PPS1D1. Methionine at the first position was not essential, whereas the positively charged Nlys at the fourth position was minimally needed, as two derivatives that were synthesized replacing this residue were almost as active as PPS1D1.     

磷脂酰丝氨酸制备的研究进展

磷脂酰丝氨酸具有改善记忆和认知能力、防治老年痴呆症、抑郁症及缓解精神头力等作用,目前在国外被用作营养补克剂广泛应用于保健食品中.着重从提取法和酶转化法2个方面综述了磷脂酰丝氨酸的制备方法,同时通过对国内外磷脂酰丝氨酸消费现状的调查,对其在食品、药品、保健品方面的应用前景进行了展望.     

“Unconventional” neutralizing activity of antibodies against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using “conventional” neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4⁺ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the “conventional” neutralization assay, do exhibit potent and broad neutralizing activity in “unconventional” ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV. Foundation item: Chinese Ministry of Science and Technology 973 program grant awarded to Paul Zhou (2006CB504308).     

＂Unconventional＂ Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However,only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes,such as high sequence diversity,heavy glycosylation,and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus,in essence the assay evaluates HIV-1 replication in CD4 T cells. Recently,several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera,although they do not have neutralizing activity when tested by the "conventional" neutralization assay,do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications,through opsonization of virus particles into macrophages and immature dendritic cells (iDCs),or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

"Unconventional" Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

Evaluation of Epstein-Barr Virus Salivary Shedding in HIV/AIDS Patients and HAART Use: A Retrospective Cohort Study

Little data is available on the evaluation of the occurrence rates of Epstein-Barr virus(EBV) in saliva and relationship with highly active antiretroviral therapy(HAART) use in HIV/AIDS patients in China. We conducted a retrospective cohort study of EBV serological tests for HIV/AIDS patients who were treated in the hospitals for infectious diseases in Wuxi and Shanghai, China from May 2016 to April 2017. The EBV-seropositive samples were identified by ELISA. EBV-specific primers and probes were used for the quantitative detection of viral DNA from saliva via quantitative real-time polymerase chain reaction. CD4 cell counts of the HIV/AIDS patients were detected by a flow cytometry. A total of 372 HIV/AIDS patients were ultimately selected and categorized for this retrospective cohort study. For EBV IgG and IgM, the HIV/AIDS HAART use(H) and non-HAART use(NH) groups had significantly higher seropositive rates than the HIV-negative control group. The HIV/AIDS(NH) group had the highest seropositive rate(IgG, 94.27%; IgM, 68.98%) and the highest incidence of EBV reactivation or infection. For salivary EBV DNA-positive rates and quantities, the HIV/AIDS(H)(73.69%) and the HIV/AIDS(NH)(100%) groups showed significantly higher values than the HIV-negative control group(35.79%,[ twofold). Further, the salivary EBV DNA-negative population had significantly higher CD4 cell counts than the EBV DNA-positive population in the HIV/AIDS(H) group and the HIV/AIDS(NH) groups. Thus, HAART use is beneficial in decreasing the EBV salivary shedding in HIV/AIDS patients and indirectly decreases EBV transmission risk.     

Metal binding to the HIV nucleocapsid peptide

 Co(II) and Zn(II) binding constants have been measured for binding to the HIV-1 nucleocapsid N-terminal metal binding domain (residues 1–18), using competition titration methods and monitoring Co(II) binding by visible absorbance spectroscopy. Enthalpies for binding were directly measured by isothermal titration colorimetry. The results are compared with recent studies of related systems, including a study of Zn(II) binding by the full length protein. Received: 1 December 1998 / Accepted: 31 December 1998     

Regulatory B cells correlate with HIV disease progression

A rare subset of IL‐10‐producing B cells, named Breg, was recently identified in mice and humans. Currently, there are no unified cell surface markers to identify Breg, and the relationship between the frequency of Breg and HIV disease progression in chronic HIV infection is unclear. In the present study, we determined whether the cell surface markers of Breg reported for other diseases are suitable for identifying Breg in HIV‐infected patients. In addition, we examined the relationship between Breg and HIV disease progression. We found that Breg frequency correlated positively with viral load and negatively with CD4 count in chronic HIV infection. Following antiretroviral treatment, the CD4 count increased and the frequency of Breg decreased stepwise. There was no difference in IL‐10 expression of CD1d^hi or CD1d^lo cells isolated from HIV‐infected patients. Therefore, CD1d may not be a marker of Breg in HIV‐infected patients.     

HIV gp41基因分段低温诱导表达

Clone N3 and C from Human immunodeficiency virus(HIV) gp41 gene were expressed using the pET expression system. When induced by IPTG at 37℃, both two clones did not express in E.coli BL21(DE)3. Howerver, when induced at 16℃, the two clones were both overexpressed, and the amount of the product was about 20% of the total bacteria protein. In Western blotting test, the protein product could react with HIV-positive serum. After IPTG induction, E. coli cells had much higher death rate at 37℃ than at 16℃; [^3H]uridine release assay also showed that after IPTG induction, E. coli had a higher release at 37℃. The results suggested that overexpression of the two proteins was due to their decreased toxicity at lower temperature.     

HIV gp41基因分段低温诱导表达

人类免疫缺陷病毒(Human immunodeficiency virus,HIV)GP41跨膜蛋白由于具有特殊的穿膜拓扑结构,对宿主菌细胞膜产生毒性作用而使其难以在E.coli中有效表达[1].本室前期工作发现GP41蛋白中有三个区域对表达菌细胞具有毒性作用,其分别为:位于N端2/3区域(N3片段:nt7373-8006)的融合肽(aa512-527)和跨膜区(Aa 684-705),它们含有丰富的疏水性氨基酸;以及位于C端1/3区域(C片段:nt8007-8339)的慢病毒裂解肽LLP1(aa 826-854)和LLP2(aa 768-788),可形成2个两亲性α螺旋,从而对宿主菌产生较强的细胞膜毒性作用使细菌大量死亡,最终致使GP41蛋白难以获得有效表达[2].为此我们尝试在低温条件(16℃)下对HIV-gp41N端2/3和C端1/3区域在大肠杆菌BL21(DE)3中进行诱导表达,并对低温条件下GP41蛋白表达对细菌细胞膜的毒性作用特征进行初步探讨.     

膜蛋白Presenilin 1的跨膜结构及催化机制

膜蛋白presenilin 1(PS1)是γ分泌酶的催化组分,是催化产生β淀粉样蛋白（β-amyloid,Aβ）的关键蛋白酶,因此也是治疗阿尔茨海默病（Alzheimer’s disease,AD）的主要靶点.PS1属于膜内裂解蛋白酶家族,这是一类在膜脂双层内部催化肽键水解断裂的蛋白酶.PS1其独特的跨膜结构和催化机制虽然还未完全揭示,但近期相关的研究取得了重要成果：PS1有10个疏水区,跨膜9次,其N端位于胞内,C端位于胞膜外或者内质网腔内,亦或不同程度地插入膜内,2个起催化作用的天冬氨酸残基都位于疏水性的膜内,膜蛋白底物被催化水解时必须先结合到酶的疏水表面上来,然后再进入位于活性部位.虽然PS1的晶体从未获得,但2006年首次解析的膜内裂解蛋白酶GlpG的晶体结构和所提出的催化机理为PS1催化机理的揭示奠定了基础,也为设计和筛选PS1/γ分泌酶的特异性抑制剂提供了理论依据.     

31P nuclear magnetic resonance studies of the association of basic proteins with multilayers of diacyl phosphatidylserine

Lysozyme, cytochrome c, poly(l-lysine), myelin basic protein and ribonuclease were used to form multilayer dispersions containing about 50% protein (by weight) with bovine brain diacyl phosphatidylserine (PS). ³¹P nuclear magnetic resonance shift anisotropies, spin-spin (T₂) and spin-lattice (T₁) relaxation times for the lipid headgroup phosphorus were measured at 36.44 MHz. At pH 7.5, lysozyme, cytochrome c, poly(l-lysine) and ribonuclease were shown to increase the chemical shift anisotropy of PS by between 12–20%. Myelin basic protein altered the shape of the phosphate resonance, suggesting the presence of two lipid components, one of which had a modified headgroup conformation. The presence of cytochrome c led to the formation of a narrow spike at the isotropic shift position of the spectrum. Of the various proteins or peptides we have studied, only poly(l-lysine) and cytochrome c had any effect on the T₁ of PS (1050 ms). Both caused a 20–30% decrease in T₁ of the lamellar-phase phosphate peak. The narrow peak in the presence of cytochrome c had a very short T₁ of 156 ms. The possibility is considered that the cytochrome Fe³⁺ contributes to the phosphate relaxation in this case. The effect of all proteins on the T₂ of the phosphorus resonance was to cause an increase from the value for pure PS (1.6 ms) to between 2 and 5 ms. The results obtained with proteins are compared with the effects of small ions and intrinsic membrane proteins on the order and motion of the headgroups of lipids in bilayers.     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and β-casein with phospholipids

The conformations adopted by β-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being α-helical and the β-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the β-casein is in a compressed state whereas the apoprotein is extended as α-helices in the plane of the interface. The chain-length dependences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC₅₀ of 200 nM with a therapeutic index of >1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. C_max and absolute bioavailability of 34S were higher and half-life and time above EC₉₅ were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.     

Electric birefringence as a means of studying the effect of anaesthetics on liposomes

Birefringence can be induced in liposome suspensions using electric fields. The fields interact predominantly with anisotropic electrical polarisabilities which give rise to induced dipole moments. Using pulsed electric fields, the optical and electrical polarisabilities and the geometrical size of the liposomes can be measured simultaneously. These parameters have been found to be very sensitive to the presence of small amounts of fluidising additives of polar and ionic nature. Illustrative data are presented for the influence of the amines ammonium chloride, methyl ammonium chloride and lignocaine and of benzyl alcohol on phosphatidylcholine/serine liposomes. Structural changes in the vesicle membranes were detected, which appeared to correlate with the biological functions, thus indicating that electric birefringence is a rapid and useful method for studying interactive phenomena in lipid membrane systems.