Nanostructure design using protein building blocks enhanced by conformationally constrained synthetic residues

Increasing efforts are being invested in the construction of nanostructures with desired shapes and physical and chemical properties. Our strategy involves nanostructure design using naturally occurring protein building blocks. Inspection of the protein structural database (PDB) reveals the richness of the conformations, shapes, and chemistries of proteins and their building blocks. To increase the population of the native fold in the selected building block, we mutate natural residues by engineered, constrained residues that restrict the conformational freedom at the targeted site and have favorable interactions, geometry, and size. Here, as a model system, we construct nanotubes using building blocks from left-handed beta-helices which are commonly occurring repeat protein architectures. We pick two-turn beta-helical segments, duplicate and stack them, and using all-atom molecular dynamics simulations (MD) with explicit solvent probe the structural stability of these nanotubular structures as indicated by their capacity to retain the initial organization and their conformational dynamics. Comparison of the results for the wild-type and mutated sequences shows that the introduction of the conformationally restricted 1-aminocyclopropanecarboxylic acid (Ac3c) residue in loop regions greatly enhances the stability of beta-helix nanotubes. The Ac3c geometrical confinement effect is sequence-specific and position-specific. The achievement of high stability of nanotubular structures originates not only from the reduction of mobility at the mutation site induced by Ac3c but also from stabilizing association forces between building blocks such as hydrogen bonds and hydrophobic contacts. For the selected synthetic residue, similar size, hydrophobicity, and backbone conformational tendencies are desirable as in the Ac3c.     

Trace element levels in fish from clean and polluted coastal marine sites in the Mediterranean Sea, Red Sea and North Sea

The bioaccumulation of Hg, Cd, Zn, Cu, Mn and Fe was evaluated in the muscle and liver tissue of four fish species (Siganus rivulatus, Diplodus sargus, Lithognatus mormyrus and Plathychtis flesus) from clean and polluted marine coastal sites in the Red Sea, Mediterranean Sea and North Sea within the framework of the MARS 1 program. Representative liver samples were screened for organic contaminants (DDE, PCBs and PAHs) which exhibited very low concentrations. The levels of Cd, Cu, Zn, Fe and Mn found in the muscle tissue in this study were similar among the four species and within the naturally occurring metal ranges. However, differences were found among the sites. In the Red Sea, Cu was higher in the muscle of S. rivulatus at Ardag and Zn at the Observatory (OBS). Cu, Zn and Mn were higher in the Red Sea than in the specimens from the Mediterranean. The differences were attributed to different diets derived from distinctively different natural environments. D. sargus from Haifa Bay (HB) had higher Cd, Cu and Mn values than specimens from Jaffa (JFA), and L. mormyrus higher Cd, Fe and Mn in HB, corresponding to the polluted environmental status of the Bay. No differences in metal levels were found among the North Sea sites, except for Fe that was lower at the Eider station. Hg was low in all the specimens, but the values varied with species and sites. The lowest Hg values were found in S. rivulatus, the herbivorous species, as expected from its trophic level. Hg in P. flesus was higher than in S. rivulatus but still low. Higher Hg values were found in the muscle tissue of L. mormyrus,with the highest values in D. sargus, both carnivorous species from the same family. Hg in D. sargus was higher in HB than in JFA, as expected, but in the larger specimens of L. mormyrus from JFA values were higher, while in the small specimens there were no differences in Hg values. The levels of all metals were higher in the liver than in the muscle, with enrichment factors ranging from 3 to 104, depending on species and sites. The lowest enrichment values were found for Hg. Based on liver values, the specimens of S. rivulatus from the OBS had the highest levels, as well as D. sargus and L. mormyrus from JFA, contrary to the known relative environmental status of the sites. Received: 25 February 1999 / Received in revised form: 5 June 1999 / Accepted: 7 June 1999     

A High-Resolution Genetic Map of the Familial Mediterranean Fever Candidate Region Allows Identification of Haplotype-Sharing among Ethnic Groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designatedMEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placedMEFVin the ∼285 kb betweenD16S468/D16S3070andD16S3376.We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placedMEFVbetweenD16S3082andD16S3373(∼200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only forD16S3370andD16S2617among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.     

REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells

REST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes.     

Horizontal transmission of the insect symbiont Rickettsia is plant-mediated

Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.     

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the “candidate gene approach” we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.     

Prostate cancer PET bioprobes: synthesis of [18F]-radiolabeled hydroxyflutamide derivatives

Approximately 80-90% of prostate cancers are androgen dependent at initial diagnosis. The androgen receptor (AR) is present in most advanced prostate cancer specimens and is believed to have a critical role in its development. Today, treatment of prostate cancer is done by inhibition of AR using antiandrogens such as flutamide (pro-drug of hydroxyflutamide), nilutamide, and bicalutamide. However, there is currently no noninvasive imaging modalities to detect, guide, and monitor specific treatment of AR-positive prostate cancer. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-propanamide [18F]-1 and N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide [18F]-2, derivatives of hydroxyflutamide, were synthesized as a fluorine-containing imaging agent candidates. A three-step fluorine-18 radiosynthesis route was developed, and the compounds were successfully labeled with a 10+/-3% decay corrected radiochemical yield, 95% radiochemical purity, and a specific activity of 1500+/-200 Ci/mmol end of bombardment (n = 10). These labeled biprobes not only may enable for the future quantitative molecular imaging of AR-positive prostate cancer using positron emission tomography but may also allow for image-guided treatment of prostate cancer.