Mass balance of heavy metal uptake by encapsulated cultures ofKlebsiella aerogenes

Dialysis was employed as a method of speciating heavy metals in cultures of an extracellular polymer forming strain ofKlebsiella aerogenes. A noncapsulated strain of the same bacterium was used as a control, and a mass balance of copper, cadmium, cobalt, nickel, and manganese in batch culture at pH 4.5 and pH 6.8 and in continuous culture at pH 6.8 was constructed. Copper and cadmium were accumulated by the cell during rapid proliferation whereas all 5 metals were bound nonspecifically by extracellular polymer produced during stationary phase and at low dilution rates. The presence of extracellular polymer appeared to inhibit cellular uptake of nickel. At the lower pH, metal uptake was considerably reduced. The results are discussed in the context of metal removal in the activated sludge process of waste water treatment.     

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Na?ve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.     

The CD16+ monocyte subset is more permissive to infection and preferentially harbors HIV-1 in vivo

HIV-1 persists in peripheral blood monocytes in individuals receiving highly active antiretroviral therapy (HAART) with viral suppression, despite these cells being poorly susceptible to infection in vitro. Because very few monocytes harbor HIV-1 in vivo, we considered whether a subset of monocytes might be more permissive to infection. We show that a minor CD16+ monocyte subset preferentially harbors HIV-1 in infected individuals on HAART when compared with the majority of monocytes (CD14highCD16-). We confirmed this by in vitro experiments showing that CD16+ monocytes were more susceptible to CCR5-using strains of HIV-1, a finding that is associated with higher CCR5 expression on these cells. CD16+ monocytes were also more permissive to infection with a vesicular stomatitis virus G protein-pseudotyped reporter strain of HIV-1 than the majority of monocytes, suggesting that they are better able to support HIV-1 replication after entry. Consistent with this observation, high molecular mass complexes of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) were observed in CD16+ monocytes that were similar to those observed in highly permissive T cells. In contrast, CD14highCD16- monocytes contained low molecular mass active APOBEC3G, suggesting this is a mechanism of resistance to HIV-1 infection in these cells. Collectively, these data show that CD16+ monocytes are preferentially susceptible to HIV-1 entry, more permissive for replication, and constitute a continuing source of viral persistence during HAART.     

Is there a role for plant-made vaccines in the prevention of HIV/AIDS?

Although educational programs have had some impact, immunization against HIV will be necessary to control the AIDS pandemic. To be effective, vaccination will need to be accessible and affordable, directed against multiple antigens, and delivered in multiple doses. Plant-based vaccines that are heat-stable and easy to produce and administer are suited to this type of strategy. Pilot studies by a number of groups have demonstrated that plant viral expression systems can produce HIV antigens in quantities that are appropriate for use in vaccines. In addition, these plant-made HIV antigens have been shown to be immunogenic. However, given the need for potent cross-clade humoral and T-cell immunity for protection against HIV, and the uncertainty surrounding the efficacy of protein subunit vaccines, it is most likely that plant-made HIV vaccines will find their niche as booster immunizations in prime-boost vaccination schedules.     

Longitudinal analysis of human immunodeficiency virus type 1 nef/long terminal repeat sequences in a cohort of long-term survivors infected from a single source

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) in a cohort of long-term survivors infected with an attenuated strain of HIV-1 acquired from a single source. Although the cohort members experienced differing clinical courses, we demonstrate similar evolution of HIV-1 nef/long-terminal repeat (LTR) sequences, characterized by progressive sequence deletions tending toward a minimal nef/LTR structure that retains only sequence elements required for viral replication. The in vivo pathogenicity of attenuated HIV-1 is therefore dictated by viral and/or host factors other than those that impose a unidirectional selection pressure on the nef/LTR region of the HIV-1 genome.     

HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism

Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.     

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.     

Brain Serotonin2 and Serotonin1A Receptors Are Altered in the Congenitally Hyperammonemic Sparse Fur Mouse

In previous studies we documented an increase in the levels of the serotonin metabolite, 5-hydroxyindoleacetic acid, in the congenitally hyperammonemic sparse fur mouse. To extend these findings, brain serotonin receptors were studied in these animals. Radioligand binding assays were performed using [3H]ketanserin to label serotonin2 sites and 8-[3H]hydroxy(di-n-propylamino)tetralin to label serotonin1A sites in cortical membrane homogenates. The capacity (Bmax) for [3H]ketanserin binding was significantly lower (-21%; p less than 0.05) in sparse fur animals than in control animals; there was no change in affinity (KD). In contrast, the capacity for 8-[3H]hydroxy(di-n-propylamino)tetralin binding was significantly greater (26%; p less than 0.05) in sparse fur compared with control animals. No difference in affinity was observed. Using two behavioral assays, the functional responsiveness of these serotonin receptors was compared in sparse fur and control animals. Head twitch activity elicited by administration of the serotonin agonist quipazine was studied as a behavior mediated by serotonin2 receptors. Compared with controls, sparse fur mice demonstrated a significantly decreased head twitch response (p less than 0.005). Hypothermia elicited by administration of 8-hydroxy(di-n-propylamino)tetralin was studied as a physiologic response mediated by serotonin1A receptors. Although there were not overall group differences in the dose-response data, there was a significant increase in the hypothermia induced by 8-hydroxy(di-n-propylamino)tetralin in sparse fur compared with control mice (p less than 0.02) at the highest dose. These data provide further support for a link between hyperammonemia and alterations in the serotonin system.