Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

Some monoclonal antibodies （mAbs） could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env^＋ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion （syncytium formation） by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.     

Structural characteristics and biological functions of the HIV-1 gp120 V3 region

Recent studies demonstrate that the V3 loop of HIV-1 gp120 plays an important role in the attachment of HIV-1 to the target cells. Several amino acids in this domain are involved in the interaction of gp120 with the co-receptors. The V3 loop elicits one of the earliest antiviral antibody responses in HIV-1 infection and has been identified as the principal neutralizing determinant (PND). A subset of antibodies to V3 loop show a broad range of neutralizing activity. Unfortunately, this loop undergoes broad mutation and is one of the hypervariable regions. Mutations of some amino acids in this PND could affect syncytium formation, virus infectivity and neutralization. Knowing the structural characteristics and biological functions of the V3 region could help us to understand mechanism of HIV infection and to develop new strategy against HIV-1. In this review, the structural characteristics, variation and biological functions of the V3 loop as well as immunological responses to the V3 loop are discussed.     

Three Candidate Peptide-Vaccines in Combination To Induce High Levels of Multiantibodies Against HIV-1

IntroductionIt has been demonstrated that humanimmunodeficiency virus type1 ( HIV- 1 ) gp41 isinvolved in HIV- 1 entry into the cell. Recentcrystal structure analysis of gp41 revealed that thebinding of gp1 2 0 to CD4and a co- receptor inducesthe conformational changes of gp41 ,resulting inthe exposure of the N- and C- domains and creationof a fusion intermediate and then the fusionprocess[1,2 ] .Itis generally agreed thatthe V3loopof gp1 2 0 plays a major role in the binding of gp1 2 0wit…     

Evidence for existence of a close association between CD14 and CXCR4 on monocytic cell line U937

The surface expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocytes can be regulated by the ligand of CD14,and the susceptibility of the cells to HIV-1 is then changed.Our previous study found that monoclonal antibody against CD14 could dramatically inhibit CXCR4-mediated chemotaxis and cell-cell fusion.Based on these studies,we explored potential relationship between CD14 and CXCR4 on monocytic cell line U937.Flow cytometry analysis showed that anti-CXCR4 monoclonal antibody (mAb) 12G5 strongly inhibited binding of the FITC-conjugated anti-CD14 monoclonal antibodies (TUK4 and UCHM1) to U937,while another CX- CR4-specific mAb B-R24 did not show any effect on this binding.On the other hand,two anti-CD14 monoclonal antibodies (TUK4 and UCH-M1) obviously inhibited the binding of the PE-conjugated anti-CXCR4 mAb 12G5 to U937 but did not inhibit the binding of mAb 12G5 to CXCR4-transfected 3T3 cells (3T3.T4.CXCR4),which indicates that the blocking of mAb 12G5 binding to CXCR4 by CD14- specific mAbs is not involved in the possibility that CD14-specific mAbs directly bind to CXCR4.These results suggested existence of a close association between CD14 and CXCR4 on monocytic cell line U937.     

Mechanism of human immunodeficiency virus type I entry into the target cells

Based on the recent advances of the research on the mechanism of HIV-1 infection, a novel model to elucidate the mechanism of HIV entry into the target cells is proposed and the perspective about the putative receptor is discussed in this review. Understanding of the crystal structure of HIV-1 transmembrane protein gp41 and the functions of HIV-1 receptor, co-receptor and the putative receptor will lead to developing effective HIV vaccine and anti-HIV drugs.     

A conserved structural motif shared by EIAV gp90 and HIV gp120

Both HIV (human immunodeficiency virus) and EIAV (equine infectious anemia virus) belong to the retroviridae family and lentivirus genus. EIAV is similar to HIV not only in its morphology, genomic structure and antigenicity, but also in its replicative and infectious characteristics. Their env genes comprise surface unit (SU) and trans- membrane protein (TM). The large number of disulfide bonds and N-glycosylation sites are prominent structural properties of SU (HIV gp120 and EIAV g…     

Molecular motions and conformational transition between different conformational states of HIV-1 gp 120 envelope glycoprotein

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations,the details of molecular motions involved in conformational transition that are crucial to intervention remain elusive. We presented comprehensive comparative analyses of the dynamics behaviors of the gp120 in its CD4-complexed,CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops by means of CONCOORD computer simulation to generate ensembles of feasible protein structures that were sub-sequently analysed by essential dynamics analyses to identify preferred concerted motions. The re-vealed collective fluctuations are dominated by complex modes of combinational motions of the rota-tion/twisting,flexing/closure,and shortness/elongation between or within the inner,outer,and bridg-ing-sheet domains,and these modes are related to the CD4 association and HIV neutralization avoid-ance. Further essential subspace overlap analyses were performed to quantitatively distinguish the preference for conformational transitions between the three states,revealing that the unliganded gp120 has a greater potential to translate its conformation into the conformational state adopted by the CD4-complexed gp120 than by the CD4-free gp120,whereas the CD4-free gp120 has a greater potential to translate its conformation into the unliganded state than the CD4-complexed gp120 does. These dynamics data of gp120 in its different conformations are helpful in understanding the relationship between the molecular motion/conformational transition and the function of gp120,and in gp120-structure-based subunit vaccine design.     

Monoclonal Anti-CD4 Antibody MT310 Binds HIV-1 gp120 Binding Site on CD4

IntroductionHuman immunodeficiency virus type1 ( HIV- 1 ) ,after binding by the envelope protein gp1 2 0 to itscellular receptor CD4,enters susceptible cellsthrough p H- independent fusion[1,2 ] . The sitewhich HIV- 1 gp1 2 0 binds is located in the firstdomain on the CD4molecule[3 5] .The anti- CD4m Ab Leu3a recognizes the gp1 2 0 binding site onCD4[6] and the anti- CD4m Ab MT31 0 stronglyinhibits HIV- 1 infection of CD4+ T cells,while theanti- CD4 m Ab MT1 5 1 weakly inhibitsinf…     

Interactions between HMG proteins (HMG1/2 and HMG14/17) and human ε-globin gene promoter (ε-promoter)

High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, thebinding of HMG proteins (HMG1/2 and HMG14/17) to the human ε-globin gene promoter (ε-promo- ter, -177-+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked ε-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome recons- titution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with ε-promoter,whi- le HMG1/2 could not. Those results indicate that the binding of HMG proteins to ε-promoter is dynamic as the nucleo- some assembling and disassembling. Wespeculated that this selective binding of HMG proteins to ε-promoter might playa critical role in the regulation of ε-globin gene expression.     

Effect of Residue Mutation on the Electrostatic Potential in EcCIC

The effect of mutation of strongly conserved porelining residues in the chloride channel EcClC on the electrostatic potential and binding free energy of the chloride ion was studied using explicit protein-membrane structures. Electrostatic potential distribution and binding free energy of the chloride ion at different binding sites in the wild-type and mutated EcClC were calculated with APBS. The potential data reveal that the electrostatic potential around the selectivity filter, especially around the site Sext and Scen becomes more negative as the residue R147 was mutated to C147. The electrostatic binding free energy shows that the binding free energy of the chloride ion at all binding sites becomes more positive as R147 was mutated. It follows that mutation of R147 decreases ion stabilization at binding sites and affects channel＇s gating.     

The effect of antibody surface packing density on its antigen binding capacity

Following our recent study of the binding of antigen hCG (human chorionic gonadotropin) onto a mouse monoclonal antibody anti-β-hCG immobilized onto the support surface, we report a more recent study of the site-specific recognition of another surface immobilized mouse monoclonal antibody anti-α-hCG by hCG. The two antibodies have similar structures and molecular weights but different site-specific recognition from hCG. They both are used in the fabrication of fertility test immunoassays. Previous study by neutron reflection has indicated the “flat-on” orientation of anti-β-hCG with its Fc and two Fabs lying flat on the support surface. The aim of this work is to determine if there is any measurable difference in hCG binding between the two antibodies that could be attributed to the steric hindrance associated with specific binding sites. The adsorption and hCG binding for anti-α-hCG were made under the surface and in solutions as for anti-β-hCG so that the outcome could be directly compared. The results show that the two antibodies are bound by hCG in an almost identical fashion, suggesting that apart from the site-specific recognition there is no measurable difference in the steric hindrance between the α and β sites.     

Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

A new mechanism of invader success： Exotic plant inhibits natural vegetation restoration by changing soil microbe community

Since the 1950s of the last century, the exotic plant, Eupatorium adenophorum, has spread rapidly across southwest China, damaging native ecosystems and causing great economic losses. We examined the pH, N, P, K, and organic matter concentrations, and the bacterial community character (by Biolog EcoPlateTM) in soils from sites heavily and lightly invaded by this exotic species. Also, soil from the lightly invaded site was treated with a water extract of E.adenophorum roots to examine the effect of the plant on soil properties. We grew three plant species, one native and two exotic, in pot experiment using soil from heavily invaded site to examine the effects of the soil on these plants growth. The soil analysis demonstrated that the pH, organic matter, total N, total P and total K in soils from the heavily invaded site were only slightly different from those of the lightly invaded site, but concentrations of NH4^ , NO3^- and available P and K in the heavily invaded site were greater than those in the lightly invaded site. The catabolic activity of soil bacterial community in the heavily invaded site was different from that in the lightly invaded site. The catabolic activity of bacterial community in soils treated by the water extract of E.adenophorum roots changed and became similar to that in soils from the heavily invaded site. The pot experiment showed that the exotic plants growth in heavily invaded soil were not different from in lightly invaded soil; however, the native plant biomass decreased dramatically when grown in soil from the heavily invaded site as compared to soil from the lightly invaded site; and the same phenomenon was found when any potential allelopathic effects by E. adenophorum were eliminated by added activated carbon to those soils. Difference in soil nutrient availability and allelopathy could not explain this phenomenon of the native plant in the soils from the heavily and lightly invaded sites. Changes observed in the soil bacterial community were obviously related to native plant growth in those tow soils. Those results suggest that changing soil microbial community may be an important part of E. adenophorum invasion process. Since the soil microbial community serves as bridge in connection of exotic and natural plants, the exotic plant could inhibit the natural plant growth and reproduction by changing the soil microbial community in invaded site.     

Neuropeptide Y receptors in a protozoaStylonychia mytilus

Radio ligand binding assays(RLBA) were used to study neuropeptide Y (NPY) receptors in a protozoa Stylonychia mytilus. The experimental results showed that 2-3×10+3/mL Stylonychia cells incubated in Pringsheim solution which contained 3H-NPY could specifically bind 3H-NPY and concomitantly present saturable characteristic. This suggested that Stylonychia possessed some specific binding sites for NPY. Scatchard transformations of binding assay for the NPY receptors at 25℃ are compatible with the specific activity of 42.47 fmol/10+3 cells and the binding equilibrium constant of 0.113 nmol/L. The data of 125I-NPY binding assay to the membrane protein extract of Stylonychia indicated that there was a significant difference between the amount of total bound and nonspecific bound of 125I-NPY. This result indicated that NPY receptors were probably localized mainly on the cell membrane.     

Molecular mechanism of the inhibitory effect of methylation outside the recognition sequence of restriction endonuclease Pvu Π on its cleavage activity

In 1991, we found that methylation outside the PvuⅡ recognition sequence could partially inhibit its cleavage activity. To clarify the molecular mechanism, three plasmids with different methylation states were constructed. Then, together with the original one, four plasmids were digested with different amounts of PvuⅡ. Results show that methylation on both sites results in 90% inhibition; moving the methylated site one base further away decreases the inhibitory effect to about 30%; with the adjacent dam methylation site eliminated, the inhibitory effect disappears. The data suggest that the inhibition of cleavage activity caused by outside methylation is not "all or none", and the degree of inhibition is dependent on the position and the number of methylated bases.     

Molecular mechanisms of serpin deficiencies

The study of serpin deficiency is currently one of the most active areas in basic medical research. Recently, three hypotheses concerning serpin deficiency have been proposed, which are referred to as the conformational disturbance hypothesis (CDH) , loop-sheet polymerisation hypothesis (LSPH) and multiple binding site hypothesis (MB-SH) . CDH was put forward to explicit serpin deficiency due to conformational change of reactive loop of serpins as a result of mutations occurring away from the reactive site residues and LSPH was to explain deficient serpins due to the formation of polymers. MBSH was proposed to explain the mechanism of the formation of stable enzyme-serpin complex via more than one binding site and blockage or mutation in any of the sites resulting in serpin deficiency. A combination of these mechanisms may be critical in understanding the roles of the many documented mutations and autoimmunities which result in qualitative and quantitative serpin deficiency.     

Mutations in Hemagglutinin of a Novel Avian-Origin H7N9 Virus That Are Critical for Receptor Binding Specificity

A novel avian-origin H7N9 influenza virus was discovered in March in China and has caused a total of131 people infected including 39 deaths in China as of June 9, 2013. Adaptation of avian viruses to efficiently infect humans requires the viral hemagglutinin（HA） binding switches from avian to human type receptors with help of some mutations in HA. As such it is critical for pandemic assessment to discover these mutations as hallmarks of adaptation. To continue our previous study of this novel H7N9 virus, we identified two sets of mutations in HA. The first set of mutations are present in the current circulating strains of 2013 H7N9 in China, and the second set are potential mutations that were found when compared to the HAs of previous human H7 subtype. These two sets of mutations exhibited unique features. The first group of mutations, on average, enhanced the HA binding to human type receptors whereas reduced that to avian types. Further the reduction of avian binding was almost three times of the increase of the human binding. The second group increased the binding to both human and avian types.But the increase in human types was almost three times of that in the avian types. Though different in their way of changing the binding preference, these two sets of mutations both contained more mutations to decrease the avian binding and increase the human binding than those that did the opposite. Our research highlighted the pandemic potential of this novel virus by showing the important mutations that could potentially help it to adapt to human hosts. Our findings offered new insights into the current state of evolution of this virus, which might be helpful for the continued surveillance of the emergence of H7N9 strains having the ability of human-to-human transmission.     

Analysis of sedimentary-geomorphologic variation and the living environment of hominids at the Shuidonggou Paleolithic site

Shuidonggou is one of the most important Upper Paleolithic sites in North China. Due to the presence of rich human remains, animal fossils, abundant sporopollen and unique geological sequence, it is the type site for Late Pleistocene to Holocene human occupation and environmental change in the Ningxia-lnner Mongolia region. Many scholars suggest that the site should be named the ＂Shuidong- gou Formation＂ of Late Pleistocene in North China. Dating results indicate that ancient human activities at the site took place 30-24 ka （Marine Isotope Stage [MIS] 3）. The climate at that time was warmer and moister than present day, and adequate precipitation led to the formation of water pack depressions where broad-leaf trees and sparse forest vegetations, as well as herbivorous animals flourished, making the area suitable for early human hunting, gathering and survival. The Neolithic human occupation happened 9-5 ka at the site, while similar environmental conditions with MIS3 occurred. The absence of human activity record in the region during the Last Glacial Maximum （MIS2） suggests that the environment was too harsh for humans to live there.