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Biomedical subjects

John P Moore

Publications and source records attributed to John P Moore.

At least 19 recordsLinked to original sources
PubMed2007

An overview of the biology of the desiccation-tolerant resurrection plant Myrothamnus flabellifolia.

BACKGROUND: Myrothamnus flabellifolia is unique as the only woody resurrection plant. It is an important plant in southern Africa because of its widespread occurrence and usage in African medicine and traditional culture. Many reports have investigated facets of its biology and the mechanisms associated with its desiccation tolerance. SCOPE: The general biology of the woody resurrection plant Myrothamnus flabellifolia is reviewed. The review focuses on the geography and ecology, systematic placement, evolution, morphology and reproductive ecology of M. flabellifolia as well as the wood anatomy and re-filling mechanism. In addition, the desiccation tolerance, ethnobotanical importance and medicinal properties of the plant are reviewed. Also, future research avenues are suggested, in particular the necessity to research the biogeography and systematics of the species and the role of the polyphenols present, as well as the molecular basis of the plant's desiccation tolerance.

Adaptation, Physiological↗
PubMed2006

HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry.

Human immunodeficiency virus type 1 (HIV-1) infection can be inhibited by small molecules that target the CCR5 coreceptor. Here, we describe some properties of clonal viruses resistant to one such inhibitor, SCH-D, using both chimeric, infectious molecular clones and Env-pseudotypes. Studies using combinations of CCR5 ligands, including small molecule inhibitors, monoclonal antibodies (MAbs) and chemokine derivatives such as PSC-RANTES, show that the fully SCH-D-resistant viruses enter target cells by using the SCH-D-bound form of CCR5. However, the way resistance to SCH-D and other small molecule CCR5 inhibitors is manifested depends on the target cell and the nature of the assay (single- vs. multi-cycle). In multi-cycle assays using primary lymphocytes, SCH-D does not inhibit resistant molecular clones, and it can even enhance their infectivity modestly. In contrast, the same viruses (as Env-pseudotypes) are significantly inhibited by SCH-D in single-cycle entry assays using U87-CD4/CCR5 cells, resistance being manifested by incomplete inhibition at high SCH-D concentrations. When a single-cycle, Env-pseudotype entry assay was performed using either U87-CD4/CCR5 cells or PBMC under comparable conditions, entry was inhibited by up to 88% in the former cells but by only 28% in the PBMC. Hence, there are both cell- and assay-dependent influences on how resistance is manifested. We also take this opportunity to correct our previous report that SCH-D-resistant isolates are also substantially cross-resistant to PSC-RANTES [Marozsan, A.J., Kuhmann, S.E., Morgan, T., Herrera, C., Rivera-Troche, E., Xu, S., Baroudy, B.M., Strizki, J., Moore, J.P., 2005. Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D). Virology 338 (1), 182-199]. A substantial element of this resistance was attributable to the unappreciated carry-over of SCH-D from the selection cultures into analytical assays.

CCR5 Receptor Antagonists↗
PubMed2006

A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120.

The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1(JR-FL). Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120-gp41 cleavage site by mutagenesis (gp140(UNC)), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1(JR-FL). All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1(JR-FL) were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes.

AIDS Vaccines↗
PubMed2006

Specific amino acids in the N-terminus of the gp41 ectodomain contribute to the stabilization of a soluble, cleaved gp140 envelope glycoprotein from human immunodeficiency virus type 1.

The HIV-1 envelope glycoprotein is expressed on the viral membrane as a trimeric complex, formed by three gp120 surface glycoproteins non-covalently associated with three membrane-anchored gp41 subunits. The labile nature of the association between gp120 and gp41 hinders the expression of soluble, fully cleaved, trimeric gp140 proteins for structural and immunization studies. Disruption of the primary cleavage site within gp160 allows the production of stable gp140 trimers, but cleavage-defective trimers are antigenically dissimilar from their cleaved counterparts. Soluble, stabilized, proteolytically cleaved, trimeric gp140 proteins can be generated by engineering an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP). We have found that SOSIP gp140 proteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared to a prototypic strain (JR-FL, subtype B). We now show that the determinants of this enhanced stability are located in the N-terminal region of KNH11144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability. The stabilized trimers retain the epitopes for several neutralizing antibodies (b12, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, suggesting that the overall antigenic structure of the gp140 protein has not been adversely impaired by the trimer-stabilizing substitutions. The ability to increase the stability of gp140 trimers might be useful for neutralizing antibody-based vaccine strategies based on the use of this type of immunogen.

Amino Acid Sequence↗
PubMed2006

Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex.

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R(2) > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.

Cell Line↗
PubMed2006

Response of the leaf cell wall to desiccation in the resurrection plant Myrothamnus flabellifolius.

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.

Cell Wall↗
PubMed2006

HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors.

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4(+) T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4(+) T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4(+) T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.

B-Cell Activating Factor↗
PubMed2006

The production of cleaved, trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein vaccine antigens and infectious pseudoviruses using linear polyethylenimine as a transfection reagent.

Trimeric HIV-1 envelope glycoproteins (Env) are now being evaluated instead of monomeric gp120 as vaccine antigens because they mimic more closely the spikes expressed on the surface of virions. Thus, it can be argued that trimers have a more native structure than gp120, so might be superior at raising neutralizing antibodies. One approach to making Env trimers is to ensure that they are cleaved at the gp120-gp41 border, but stabilized by other, engineered substitutions such as intra-subunit disulfide bonds (SOS and SOSIP gp140 proteins). However, the production of properly folded, cleaved trimers is complicated by the requirement for co-expression of the exogenous protease furin, to facilitate the efficient processing oft the gp120-gp41 cleavage site. Also, yields of purified trimeric SOSIP gp140 proteins are usually moderate and for scale-up procedures the cost of transfection reagents becomes an important economical factor. Here, we assess the optimal culture conditions for the transient expression of these complex proteins. We found that the use of linear polyethylenimine 25 kDa (PEI25k) as a transfection aid was a cost-efficient, economical alternative to several commercially available products. By using PEI25k and an optimized plasmid:furin ratio, we could express proteolytically mature, trimeric Env vaccine antigens at levels high enough for use in immunization or structural studies. We also show that the same transfection method can be used to generate infectious pseudoviruses.

AIDS Vaccines↗
PubMed2006

Interaction of small molecule inhibitors of HIV-1 entry with CCR5.

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands.

Amides↗
PubMed2006

Construction and characterization of soluble, cleaved, and stabilized trimeric Env proteins based on HIV type 1 Env subtype A.

The generation of an antibody response capable of neutralizing a broad range of clinical isolates remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Envelope glycoprotein (Env)-based vaccine candidates will also need to take into account the extensive genetic diversity of circulating HIV-1 strains. We describe here the generation of soluble, stabilized, proteolytically cleaved, trimeric forms of Env (SOSIP gp140 proteins) based on contemporary Env subtype A viruses from East Africa. We discuss issues associated with the construction, purification, and characterization of such complex proteins; not all env sequences allow the expression of trimeric proteins. However, stabilized trimers from one such protein, KNH1144 SOSIP gp140, were successfully made. These proteins are now being prepared for preclinical immunogenicity studies.

AIDS Vaccines↗
PubMed2005

The South African and Namibian populations of the resurrection plant Myrothamnus flabellifolius are genetically distinct and display variation in their galloylquinic acid composition.

The polyphenol contents and compositions in desiccated leaves of Myrothamnus flabellifolius plants collected in various locations in Namibia and South Africa were analyzed using UV spectroscopy and high-performance liquid chromatography-mass spectrometry. A study of the genetic relatedness of these populations was also performed by determination of the DNA sequence of the intergenic spacer region between the psbA and the trnH genes in the chloroplast genome. Namibian M. flabellifolius plants contained significantly more polyphenols than South African plants. Namibian plants essentially contained a single polyphenol, 3,4,5-tri-O-galloylquinic acid, whereas South African plants contained a variety of galloylquinic acids including 3,4,5-tri-O-galloylquinic acid together with higher molecular weight galloylquinic acids. Sequence analysis revealed a 1.4% divergence between Namibian and South African plants corresponding to the separation of these populations of approximately 4 x 10(6) years. The significance of the poly-phenol content and composition to the desiccation tolerance of the two populations is discussed.

Base Sequence↗
PubMed2005

Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor.

Pre-exposure oral prophylaxis with antiviral drugs is a potential method for preventing transmission of human immunodeficiency virus type 1 (HIV-1). We show that oral delivery of CMPD167, a small molecule that binds to the CCR5 coreceptor, for 10-14 d can protect a substantial proportion of macaques from vaginal infection with a CCR5-using virus (SHIV-162P3). The macaques that became infected despite receiving CMPD167 had reduced plasma viremia levels during the earliest stages of infection.

Administration, Oral↗
PubMed2005

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion.

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.

Administration, Intravaginal↗
PubMed2005

Use of alternate coreceptors on primary cells by two HIV-1 isolates.

Two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors on transfected cell lines were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. The R5 isolate grew in Delta32/Delta32 CCR5 PBMC in the absence or presence of AMD3100, a CXCR4-specific inhibitor, indicating that it uses a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6, CCR3, and CCR8 on transfected cells and was able to replicate in the absence or presence of AMD3100 in Delta32/Delta32 CCR5 PBMC. It was insensitive to eotaxin, vMIP-1 and I309 when tested individually, but was inhibited completely when vMIP-1 or I309 was combined with AMD3100. Both I309 and vMIP-1 bind CCR8, strongly suggesting that this isolate can use CCR8 on primary cells. Collectively, these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry.

Adult↗
PubMed2005

Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D).

We describe the generation of two genetically related human immunodeficiency virus type 1 (HIV-1) isolates highly (>20,000-fold) resistant to the small molecule CCR5 inhibitor, SCH-417690 (formerly SCH-D). Both viruses were cross-resistant to other small molecules targeting entry via CCR5, but they were inhibited by some MAbs against the same coreceptor on primary CD4+ T-cells. The resistant isolates remained sensitive to inhibitors of other stages of virus entry, and to replication inhibitors acting post-entry. Neither escape mutant could replicate detectably in peripheral blood mononuclear cells (PBMC) from two donors homozygous for the CCR5-Delta32 allele and both were insensitive to the CXCR4-specific inhibitor, AMD3100. Hence, the SCH-D escape mutants retained the R5 phenotype. One of the resistant isolates was, however, capable of replication in U87.CD4.CXCR4 cells and, after expansion in those cells, was sensitive to AMD3100 in primary CD4+ T-cells. Hence, some X4 variants may be present in this escape mutant swarm. A notable observation was that the SCH-D escape mutants were also cross-resistant to PSC-RANTES and AOP-RANTES, chemokine derivatives that are reported to down-regulate cell surface CCR5 almost completely. However, the extent to which CCR5 is down-regulated was dependent upon the detection MAb. Hence, the escape mutants may be using a CCR5 configuration that is only detected by some anti-CCR5 MAbs. Finally, two SCH-D-resistant clonal viruses revealed no amino acid changes in the gp120 V3 region relative to the parental viruses, in marked contrast to clones resistant to the AD101 small molecule CCR5 inhibitor that possess 4 such sequence changes. Several sequence changes elsewhere in gp120 (V2, C3 and V4) were present in the SCH-D-resistant clones. Their influence on the resistant phenotype remains to be determined.

Alleles↗