Isolation of human monoclonal antibodies that neutralize human rotavirus

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 micro g/ml.     

Isolation of human sequences that replicate autonomously in human cells.

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.     

Heat-inducible human factor that binds to a human hsp70 promoter.

A factor found in nuclear extracts of human cells bound to the heat shock element of a human heat shock protein 70 gene. The level of this factor was significantly increased after heat shock. This induction was rapid and was not blocked by cycloheximide, suggesting that an initial event in the response of a human cell to heat is the activation of a preexisting regulatory factor.     

Polypeptides that inhibit human breast cancer cell division

Medium sized (10-15 amino acids) polypeptides isolated from normal human urine inhibit the growth of five established cell lines of human mammary cancer. Cytostatic and cytotoxic effects are dose dependent and reversible at lower doses. Evidence suggests a block to cell division in S phase.     

Sex-specific demographic behaviours that shape human genomic variation

In the human species, the two uniparental genetic systems (mitochondrial DNA and Y chromosome) exhibit contrasting diversity patterns. It has been proposed that sex-specific behaviours, and in particular differences in migration rate between men and women, may explain these differences. The availability of high-density genomic data and the comparison of genetic patterns on autosomal and sex chromosomes at global and local scales allow a reassessment of the extent to which sex-specific behaviours shape our genome. In this article, we first review studies comparing the genetic patterns at uniparental and biparental genetic systems and assess the extent to which sex-specific migration processes explain the differences between these genetic systems. We show that differences between male and female migration rates matter, but that they are certainly not the only contributing factor. In particular, differences in effective population size between men and women are also likely to account for these differences. Then, we present and discuss three anthropological processes that may explain sex-specific differences in effective population size and thus human genomic variation: (i) variance in reproductive success arising from, for example, polygyny; (ii) descent rules; and (iii) transmission of reproductive success.     

Monoclonal antibodies that distinguish between subspecies of human interferon-alpha and that detect interferon oligomers

Monoclonal antibodies to human interferons (HuIFN) of the alpha-class have been prepared by screening against 125I-labeled IFN in a rapid liquid-phase radioimmunoassay. All of the six antibodies produced react with HuIFN-alpha 2 and with some components of HuIFN-alpha N (Namalwa); three of the antibodies also bind HuIFN-alpha 1, and these either do not bind or bind very weakly the 25K component of Namalwa. Reaction of the antibodies with IFN components blotted onto nitrocellulose after separation on reducing gels suggests that two of the antibodies are against conformational determinants, whereas the epitopes recognized by the other antibodies are not destroyed by reduction or SDS treatment; these antibodies can be used to detect the presence of oligomers in IFN preparations. From the reaction of the antibodies with different alpha-IFN in immunoblots, in an antiviral assay, and in an ELISA, it was concluded that at least five different epitopes are recognized by the six antibodies, only one of which is non-neutralizing.     

A human enzyme that liberates uracil from DNA

An enzyme activity which acts specifically on uracil-containing DNA was found in human placenta and cultured fibroblasts. The enzyme liberates uracil from DNA in the presence of EDTA at pH 7.5. Almost equal levels of the activity were found in normal and xeroderma pigmentosum cell lines (complementation group A).     

Determining molecular forces that stabilize human aquaporin-1

Atomic force microscopy (AFM) was used to measure the forces stabilizing human aquaporin-1 (hAQP1), a tetrameric transmembrane protein that forms highly specific water channels. To this end, the AFM tip was attached to the C-terminus of hAQP1 and secondary structure elements were extracted from the membrane while the single-molecule force-extension curve was being recorded. Force peaks, reflecting the unfolding of secondary structure elements, could be interpreted in depth using the atomic model of hAQP1. Different classes of force-extension curves indicated the existence of alternative unfolding pathways for individual proteins. In addition, transmembrane helices at the periphery of the hAQP1 tetramer exhibited smaller extraction forces than helices at the interface between hAQP1 monomers. These results represent the first direct assessment of intermolecular forces defining the oligomeric state of a membrane protein.     

Natural product coumarins that inhibit human carbonic anhydrases

Natural products (NPs) have proven to be an invaluable source of new chemotherapies yet very few have been explored to source small molecule carbonic anhydrase (CA) inhibitors. CA enzymes underpin physiological pH and are critical to the progression of several diseases including cancer. The present study is the first to more widely investigate NP coumarins for CA inhibition following the recent discovery of a NP coumarin CA inhibitor. We assembled a NP library comprising 24 plant coumarins (compounds 4–27) and three ascidian coumarins (compounds 28–30) that together provide a diverse collection of structures containing the coumarin pharmacophore. This library was then evaluated for inhibition of six human CA isozymes (CAs I, II, VII, IX, XII and XIII) and a broad range of inhibition and isozyme selectivity profiles were evident. Our findings provide a platform to support further evaluation of NPs for the discovery of new chemotypes that inhibit disease relevant CA enzymes.     

Standardization of factors that influence human urine metabolomics

In nutritional metabolomics a large inter- and intra-subject variability exists, and thus, it becomes important to limit the variance introduced by external factors. In a composite controlled study with full provision of all food for the standardized intervention, human urinary metabolite profiles were investigated for different factors, such as handling of urine collections, diet standardization, diet culture, cohabitation and gender. In study A, 8 healthy subjects (4 men; 4 women) collected 24-h urine, splitting each void into two specimens stored either at 4°C or at room temperature. In study B, 16 healthy subjects (7 men; 9 women) collected 24-h urine for three days while being on a standardized diet. Samples were analyzed by ¹H NMR and chemometrics. The NMR profiles indicated the presence of metabolites presumably originating from bacterial contamination in 3 out of 16 sample collections stored at room temperature. On the contrary, no changes in the NMR profiles due to contamination occurred in the 24-h urine samples stored at 4°C. The study also showed a trend towards a reduced inter- and intra-individual variation during 3 days of diet standardization. In study A, the urine metabolome showed a clear effect of diet culture and cohabitation, but these effects significantly attenuated after diet standardization (study B). Besides, gender-specific differences were found in both studies. Our results emphasize that best practice for any metabolomic study is a standardized, chilled sample collection procedure, and recommend that diet standardization is performed prior to dietary interventions in order to reduce intra- and inter-subject variability.     

子宫颈癌预防用人乳头瘤病毒疫苗及其展拓

人乳头瘤病毒(HPV)是人子宫颈癌病原体,这一发现奠定了子宫颈癌预防用人乳头瘤病毒疫苗得以问世的科学基础。临床实践证明,默克(Merck)公司的Gardasil和葛兰素史克(GSK)公司的Cervarix这两种疫苗可预防由HPV16和HPV18引起的子宫颈癌,有效率几乎达100%。Gardasil和Cervarix的成功激动了围绕Gardasil和Cervar-ix的展拓研究。     

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified. Two MAbs, 2E1 and 3D11, recognized an epitope in the amino-terminal 689 residues of apoB in native apoB-containing lipoproteins (LpBs) from human plasma or from the supernatant of human hepatoma HepG2 cells, but did not react with LpB from HuBTg+/+ mice or LpB secreted by human apoB-100-transfected rat McArdle 7777 hepatoma cells. 2E1 reacted weakly and 3D11 reacted strongly with apoB from HuBTg+/+ mice after SDS-PAGE. The lack of expression of the 2E1 and 3D11 epitopes on native LpB from HuBTg+/+ mice did not solely reflect the abnormal lipid composition of murine LpB. Both epitopes were detected in all human plasma samples tested and in all human plasma LpB classes. Therefore, human apoB expressed by rodent hepatocytes or hepatoma cells appears to adopt a different conformation or undergoes different posttranslational modification than apoB expressed in human hepatocytes or hepatoma cells.     

The search for genotypes that underlie human performance phenotypes

For a species spread throughout the world, humans are remarkably invariant; yet there has always been more interest in the slight differences between individuals than in the great commonality. This is especially true in athletic endeavours, where nearly immeasurable differences in performance can separate the winner from the rest of the competitors. There is little doubt that performance is influenced by environment, as the effects of diet and training on athletic ability have long been known, if not completely understood; however, the contribution of an individual's genetic make-up is less clear. The dominance of particular nationalities, ethnic groups, or families in various sporting events is often perceived as evidence that heritage (biological or cultural), plays a role in the development of athletic skills. Further complicating the issue are the interactions between genetic background and environment, as both of these fundamental arbiters of development rarely act independently. Despite the complexity of the problem, numerous researchers have attempted to elucidate the effects of genetic background on physical performance and, more recently, to identify the specific genetic variants that contribute to performance. This article reviews some of these studies with a focus on the methodologies employed.     

A defined human system that supports bidirectional mismatch-provoked excision

Mismatch-provoked excision directed by a strand break located 3' or 5' to the mispair has been reconstituted using purified human proteins. While MutSalpha, EXOI, and RPA are sufficient to support hydrolysis directed by a 5' strand break, 3' directed excision also requires MutLalpha, PCNA, and RFC. EXOI interacts with PCNA. RFC and PCNA suppress EXOI-mediated 5' to 3' hydrolysis when the nick that directs excision is located 3' to the mispair and activate 3' to 5' excision, which is dependent on loaded PCNA and apparently mediated by a cryptic EXOI 3' to 5' hydrolytic function. By contrast, RFC and PCNA have only a limited effect on 5' to 3' excision directed by a 5' strand break.     

Dendrimer-based BH3 conjugate that targets human carcinoma cells

Our previous studies have demonstrated the efficacy of generation 5 poly(amidoamine) dendrimers (G5-PAMAM) as a platform for the targeted delivery of chemotherapeutics. However, anticancer therapy can be subverted by anti-apoptotic changes in cancer cells. Bcl-2 and several of its peptides are commonly overexpressed in a number of cancers. A means to reverse this anti-apoptotic mechanism is to expose the cells to BH3 peptide, which has a homology to the anti-apoptotic Bcl-2 protein. This cannot be done indiscriminately because it can induce apoptosis in normal cells. In order to specifically target BH3 peptides to cancer cells, we synthesized a trifunctional, G5-PAMAM-based nanodevice to which folic acid was conjugated as a targeting agent, along with fluorescein isothiocyanate as the reporter agent and BH3 peptides that were used to induce apoptosis. The results show, for the first time, the therapeutic potential of targeted BH3 peptides as a means of inducing apoptosis by interfering with anti-apoptotic proteins within specific cells.     

The neural systems that mediate human perceptual decision making

Perceptual decision making is the act of choosing one option or course of action from a set of alternatives on the basis of available sensory evidence. Thus, when we make such decisions, sensory information must be interpreted and translated into behaviour. Neurophysiological work in monkeys performing sensory discriminations, combined with computational modelling, has paved the way for neuroimaging studies that are aimed at understanding decision-related processes in the human brain. Here we review findings from human neuroimaging studies in conjunction with data analysis methods that can directly link decisions and signals in the human brain on a trial-by-trial basis. This leads to a new view about the neural basis of human perceptual decision-making processes.     

Natural selection on genes that underlie human disease susceptibility

What evolutionary forces shape genes that contribute to the risk of human disease? Do similar selective pressures act on alleles that underlie simple versus complex disorders [1-3]? Answers to these questions will shed light onto the origin of human disorders (e.g., [4]) and help to predict the population frequencies of alleles that contribute to disease risk, with important implications for the efficient design of mapping studies [5-7]. As a first step toward addressing these questions, we created a hand-curated version of the Mendelian Inheritance in Man database (OMIM). We then examined selective pressures on Mendelian-disease genes, genes that contribute to complex-disease risk, and genes known to be essential in mouse by analyzing patterns of human polymorphism and of divergence between human and rhesus macaque. We found that Mendelian-disease genes appear to be under widespread purifying selection, especially when the disease mutations are dominant (rather than recessive). In contrast, the class of genes that influence complex-disease risk shows little signs of evolutionary conservation, possibly because this category includes targets of both purifying and positive selection.     

Triterpene derivatives that inhibit human immunodeficiency virus type 1 replication

Triterpene derivatives were analyzed for anti-HIV-1 activity and for cellular toxicity. Betulinic aldehyde, betulinic nitrile, and morolic acid derivatives were identified to have anti-HIV-1 activity. These derivatives inhibit a late step in virus replication, likely virus maturation.