Conjugated platinum(IV)-peptide complexes for targeting angiogenic tumor vasculature

The integrins alpha vbeta3 and alpha vbeta5 and the membrane-spanning surface protein aminopeptidase N (APN) are highly expressed in tumor-induced angiogenesis, making them attractive targets for therapeutic intervention. Both integrins and APN recognize a broad range of peptides containing RGD (Arg-Gly-Asp) and NGR (Asn-Gly-Arg) motifs, respectively. Here, we describe the design, synthesis, and characterization of a series of mono- and difunctionalized platinum(IV) complexes in which a conjugated peptide motif, containing RGD, (CRGDC)c, (RGDfK)c, or NGR, is appended as a "tumor-homing device" to target tumor endothelial cells selectively over healthy cells. Platinum(IV)-peptide complexes with nonspecific amino acids or peptide moieties were prepared as controls. Concentration-response curves of these compounds were evaluated against primary proliferating endothelial cells and tumor cell lines and compared to those of cisplatin, a well-described platinum-based chemotherapeutic agent. The Pt(IV)-RGD conjugates were highly and specifically cytotoxic to cell lines containing alpha vbeta3 and alpha vbeta5, approaching the activity of cisplatin. The Pt(IV)-NGR complexes were less active than Pt(IV)-RGD-containing compounds but more active than nonspecific Pt-peptide controls. Integrin alpha vbeta3 mediated, at least in part, the anti-proliferative effect of a Pt(IV)-RGD conjugate, as demonstrated by a decreased inhibitory response when endothelial cells were either (1) incubated with an excess of alpha vbeta3/alpha vbeta5-specific RGD pentapeptides or (2) transfected with RNAi for beta 3, but not beta 1, integrins. These results suggest a rational approach to improved chemotherapy with Pt(IV)-peptide conjugates by selective drug delivery to the tumor compartment.     

Isolation and characterization of 12 polymorphic microsatellite markers in the tropical house gecko (Hemidactylus mabouia)

We present 12 variable microsatellite loci isolated from the invasive tropical house gecko, Hemidactylus mabouia. Polymerase chain reaction (PCR) primers were tested on 39 individuals from two locations in Miami, Florida. Heterozygote deficiency was detected for four loci, and we attribute this to possible null alleles or population substructure. Some loci successfully amplified PCR products in several congeners, indicating their potential for use in other geckos.     

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.     

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia

We previously demonstrated prolonged, profound CD4⁺ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3–4 months. Both cohorts produced naïve T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4⁺ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.     

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

A Revision of Australian River Prawns, Macrobrachium (Crustacea: Decapoda: Palaemonidae)

A taxonomic revision of Australian Macrobrachium identified three species new to the Australian fauna – two undescribed species and one new record, viz. M. auratumsp. nov., M. koombooloombasp. nov., and M. mammillodactylus(Thallwitz, 1892). Eight taxa previously described by Riek (1951) are recognised as new junior subjective synonyms, viz. M. adscitum adscitum, M. atactum atactum, M. atactum ischnomorphum, M. atactum sobrinum, M. australiense crassum, M. australiense cristatum, M. australiense eupharum of M. australienseHolthuis, 1950, and M. glypticumof M. handschiniRoux, 1933. Apart from an erroneous type locality for a junior subjective synonym, there were no records to confirm the presence of M. australe(Guérin-Méneville, 1838) on the Australian continent. In total, 13 species of Macrobrachiumare recorded from the Australian continent. Keys to male developmental stages and Australian species are provided. A revised diagnosis is given for the genus. A list of 31 atypical species which do not appear to be based on fully developed males or which require re-evaluation of their generic status is provided. Terminology applied to spines and setae is revised.     

Isolation and Phylogenetic Analysis of Novel Viruses Infecting the Phytoplankton Phaeocystis globosa (Prymnesiophyceae)

Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.