N A N O P R O B E S     E - N E W S

Vol. 7, No. 1          January 18, 2006

It is well established that all metazoan phyla, including the Porifera (sponges), evolved from a common ancestor, yet they have diverse body plans of different complexity. Therefore, finding the common, basic principles of pattern formation in all phyla is of great interest. All metazoan body plans include the formation of at least one axis that runs from the apical to the basal region; examples include the Porifera and the Cnidaria (diploblastic animals). One possible basis for the formation of the Bauplan in sponges is the construction of their skeleton by spicules. In Demospongiae and Hexactinellida, these spicules consist of silica; their formation as the building blocks of the skeleton begins with the expression of an enzyme termed silicatein. Müller and co-workers have investigated this process microscopically, using the model species Suberites domuncula.

Spicule formation is a rapid process; they can grow up to 5 microns per hour, and the study of their genesis is difficult in intact organisms. Therefore, spicule development was studied in an in vitro cell culture system, the primmorphs. For direct examination by transmission electron microscopy, sponge samples were cut into pieces (2 mm³), incubated in 0.1 M phosphate buffer (supplemented with 2.5% glutaraldehyde; 0.82% NaCl, pH 7.4), and washed in 0.1 M phosphate buffer (1.75% NaCl) at room temperature. After treatment with 1.25% NaHCO₃, 2% OsO₄, and 1% NaCl, the samples were dehydrated with ethanol, incubated with propylene oxide, fixed in propylene oxide/Araldite (2:1), covered with pure Araldite, and hardened at 60°C for 2 days. Blocks were cut into 60-nm ultrathin slices, transferred onto coated copper grids, and analyzed using a Tecnai 12 microscope (FEI Electron Optics). Immunogold labeling was performed on tissue samples treated in 0.1% glutaraldehyde / 3% paraformaldehyde buffered in 0.1 M phosphate buffer at pH 7.4; after 2 h, the material was dehydrated in ethanol and embedded in LR-White Resin. 60 nm slices were cut, blocked with 5% bovine serum albumin (BSA) in PBS and then incubated with a primary polyclonal antibody (PoAb) against the purified filaments from spicules, raised in female rabbits (PoAb-aSilic: 1 : 1,000) for 12 hours at 4°C. In controls, pre-immune serum was used. Sections were then washed three times with PBS containing 1% BSA, then incubated with a 1 : 100 or 1 : 200 dilution of Nanogold anti-rabbit IgG for 2 hours. Sections were then rinsed in PBS, treated with 1% glutaraldehyde / PBS for 5 minutes, washed and dried, and then subsequently silver enhanced using the procedure described by Danscher. Samples were examined with the Tecnai 12 microscope.

Spicule growth begins intracellularly around an axial filament composed of silicatein. When the first layer of silica is made, the spicules are extruded from the cells and completed extracellularly to reach their final form and size. At the initial stage, the axial canal is composed only of silicatein, whereas later, membranous structures and fibrils (1015 nm in width) can also be identified, suggesting that intracellular components protrude into the axial canal. Most immunogold labeling is associated with the surface of the spicule and axial filament; at higher magnification, it can be clearly associated with the inner and outer surface of the spicule. Strong immunogold staining of the axial filament in the axial canal was also found. Antibodies against silicatein were also applied for Western blotting: this showed that intracellular silicatein is processed to the mature form (24 kDa), whereas the extracellular form is the pro-enzyme with the propeptide (33 kDa). Immunohistological analysis showed that silicatein exists in the axial canal (axial filament) and on the surface of the spicules, suggesting that they grow by apposition. The enzymatic reaction of silicatein was found to be inhibited by anti-silicatein antibodies, confirming that the 24kDa form is the active enzyme. These data provide, for the first time, a comprehensive outline of spicule formation.

While the first steps of spicule formation within the cells are becoming clear, the silicatein-specific antibodies were also used with Nanogold labeling to determine whether silicatein exists in the bulky extracellular space in adult specimens. In the mesohyl compartment strings, which are decorated with Nanogold, were readily identified, and were especially dense around the area of spicule formation. At a higher power the strings were found to be regularly arranged and to follow the surface shape of the spicule. Especially dense were the strings at the ends of the spicules, where the longitudinal growth of the spicules proceeds; one possibility is that extracellular collagen might be involved in the organization of strings consisting mainly of silicatein, but the process by which the extracellular silicatein strings are formed remains to be studied. Understanding this morphogenetic process in particular will give us an insight into the construction of the highly diverse skeletons of the siliceous sponges, which evolved between two periods of glaciations, the Sturtian glaciation (710680 MYA) and the Varanger-Marinoan ice ages (605585 MYA). Sponges can be considered a kind of living fossil, preserving evolutionary trends which remained unique in the metazoan kingdom.

References:

• Müller, W. E. G.; Belikov, S. I.; Tremel, W.; Perry, C. C.; Gieskes, W. W. C.; Boreiko, A., and Schroder, H. C.: Siliceous spicules in marine demosponges (example Suberites domuncula). Micron, 37, 107-120 (2006).
  • Abstract (courtesy of Science Direct).

• Müller, W. E. G.; Rothenberger, M.; Boreiko, A.; Tremel, W.; Reiber, A., and Schröder, H. C. Formation of siliceous spicules in the marine demosponge Suberites domuncula. Cell Tissue Res., 321, 285297 (2005).
  • Abstract (courtesy of SpringerLink).

More information:

• Nanogold conjugates: Catalog information
• Nanogold-antibody conjugates: Product information
• Technical help: Nanogold conjugates
• References: Nanogold labeling

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanogold® Labeling of Plasmids

If you are labeling a synthetic oligonucleotide, these modifications are readily introduced during oligonucleotide synthesis using modified phosphoramidites such as those from Glen Research. However, if you are working with a plasmid or other enzymatically-generated or naturally occurring oligonucleotide, you can't use modified phosphoramidites: so, what are your options? Actually, there are several, and we describe some possible approaches below.

• Use a modified nucleotide during plasmid preparation. A variety of modified bases are available that can be incorporated by polymerases or other enzymes. In a previous article, we reported how Willner and group used a 10 : 1 mixture of unmodified dUTP with an amino-modified form of dUTP, 5-[3-Aminoallyl]-2'-deoxyuridine 5'-triphosphate (amino-dUTP) to prepare amino-modified DNA in cancer cells. This provides a primary aliphatic amine, which may be labeled using Mono-Sulfo-NHS-Nanogold. Malecki used nick translation with a biotin-conjugated dUTP to incorporate biotin; the biotinylated DNA was then incubated with Nanogold-streptavidin as part of the transfection complex. Boublik and co-workers used 2-thiocytidine. If you incorporate a small amount of one of these into your plasmid preparation mixture, it will be incorporated and the reactive groups will be introduced. Trilink Biotechnologies offer a wide range of modified and functionalized nucleotides that may be suitable for this type of modification. The strategy is shown in Scheme 1:

  

  Incorporation of 5-[3-Aminoallyl]-2'-deoxyuridine 5'-triphosphate (amino-dUTP) into plasmid, followed by labeling with Mono-Sulfo-NHS-Nanogold at an incorporated amine site.

  If present in small proportions (for example, if about 20% of the nucleotide is in the modified form) will be incorporated with relatively low frequency into the plasmid, facilitating lower-density labeling which has less likelihood of affecting transfection. The actual proportion of modified nucleotide that you use will be determined by the size of the construct you are preparing and the number of labeling sites you wish to introduce.

• Use a photoreactive cross-linker to introduce a reactive site or a hapten into the completed plasmid. A good source of cross-linking reagents with a wide variety of functionalities is Pierce, who provide a wide range of different types of reactivity, functionality and cleavability. Pierce also provides an online cross-linker selection guide that you can use to identify the best cross-linkers for your application. This tool lets you specify the reactivities and functionalities to be introduced; select "non-selective/photoreactive" for "Functional Group Reactive Toward 1" and "Amine" for "Functional Group Reactive Toward 2;" this will provide you with a list of cross-linkers that you can use to introduce amines. If you then also select "Cleavable by thiols" under "cleavability," the reagents you select will include a disulfide within the chain, or an alternative group which, when cleaved, provides a thiol suitable for labeling with Monomaleimido Nanogold.

  Another example of this approach is the Fast-tag system from Vector Laboratories; this provides another method for introducing thiols which can then be labeled with Monomaleimido Nanogold. An alternative supplier of novel cross-linking reagents is Molecular Biosciences; this company also sorts its products by reactivity.

  You can also use this approach to biotinylate the oligonucleotide, using a photoreactive biotinylation reagent. The biotinylated plasmid is then localized with Nanogold-streptavidin. This reagent provides high sensitivity and specificity for in situ hybridization; it is described in full, with references, links and full protocols, in a special report on our web site.

• Use Positively Charged Nanogold, which binds to the negatively charged oligonucleotide backbone, to selectively decorate the plasmid. This approach has been used to decorate DNA for STEM visualization, and when used with linear DNA, provides a potential method for preparing DNA-based conductive nanowires. In our paper from Microscopy and Microanalysis 2001, we have described the preparation of labeled DNA by incubating the DNA with a solution of Positively Charged Nanogold. The reaction is straightforward: positively charged Nanogold may be mixed with double-stranded DNA either with the DNA immobilized on a grid, or in solution, and it will bind through the interaction of the positive charges on the Nanogold with the negative charges on the DNA backbone. A near neutral to slightly acidic pH (6 to 7.5) may work best. Unbound Nanogold may be removed either by washing the labeled DNA when it is bound to a support or a surface, or by chromatographically separating the labeled DNA using gel filtration over a column such as Amersham Pharmacia Superose-6 or Superose-12. Some trial-and-error adjustment of the ratio of Nanogold to DNA may be necessary if the reaction is carried out in solution: some precipitation may be observed if the Nanogold binds to more than one DNA molecule, but this may be eliminated by increasing the excess of Nanogold.

References:

• Functionalization of plasmids:

  Malecki, M.: Preparation of plasmid DNA in transfection complexes for fluorescence and spectroscopic imaging. Scanning Microsc. Suppl. (Proc. 14th Pfefferkorn Conf.); Malecki, M., and Roomans, G. M. (Eds.). Scanning Microscopy International, Chicago, IL, 10, 1-16 (1996).

• Functionalization and undecagold labeling of tRNA:

  Hainfeld, J. F.; Sprinzl, M.; Mandiyan, V.; Tumminia, S. J., and Boublik, M.: Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster. J. Struct. Biol., 107, 1-5 (1991).

  • Abstract (courtesy of Science Direct).

More information:

• Nanogold labeling reagents: Catalog information
• Technical help: Nanogold labeling reagents
• DNA Nanowires: our paper from Microscopy & Microanalysis 2001
• Technical help: Nanogold labeling of oligonucleotides
• Nanogold-streptavidin: product information

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

High Resolution Negative staining with NanoVan and Nano-W

The ideal negative stain is completely amorphous, since any crystallization can obscure features of interest. When combining negative staining with ultrastructural gold labeling, it is helpful if the negative stain is not too electron-dense, so that it does not make the gold difficult to see, and the overall contrast allows easy visualization of both the gold label and the negative stain. NanoVan and Nano-W are two novel negative staining reagents from Nanoprobes, based on vanadium and tungsten respectively, that allow negative staining with a range of different densities. NanoVan is recommended for use with Nanogold® because the lower atomic number of vanadium means that the stain is less electron-dense than heavy metal-based stains such as uranyl acetate or lead citrate: it is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels.

Advantages of these reagents:

• NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
• Near-neutral pH results in better ultrastructural preservation.
• NanoVan is less susceptible to electron beam damage than uranyl acetate.
• Fine grain allows high imaging resolution.

New results obtained with these reagents appear in three recent papers. In the first two, Kirkham and co-workers describe the identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin (Journal of Clinical Microbiology), and the construction and immunological characterization of a novel nontoxic protective pneumolysin mutant for use in pneumococcal vaccines (Infection and Immunity).

Streptococcus pneumoniae (the pneumococcus) is the predominant cause of fatal infections such as bacterial pneumonia and meningitis. Pneumococci can be divided into 90 serotypes depending on the immunochemistry of their capsular polysaccharide; less than 20% are the major cause of disease. Serotype 1 S. pneumoniae has remained one of the most prevalent invasive serotypes, with a high prevalence of serotype 1 disease throughout Europe, South America, Africa, and Asia. Serotype 1 is associated with complicated pneumonia, pulmonary empyema, peritonitis, and salpingitis and has been directly linked to mortality, irrespective of factors such as age, environment, and leukocyte count of patients. Serotype 1 is also associated with a higher ratio of hospitalization versus ambulatory care compared with pneumococcal infections from other serotypes. Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is ranked in the top five most prevalent pneumococcal serotypes in at least 10 countries. Although the recently licensed seven-valent polysaccharide conjugate vaccine (7PCV) is highly efficacious against pneumococcal disease caused by the seven vaccine serotypes, the decrease observed with otitis media from serotypes included in 7PCV has coincided with a marked increase in disease caused by non-vaccine serotypes. Capsule polysaccharide from serotype 1 pneumococci is not included in the licensed 7PCV, and with the rise in non-vaccine serotypes, this situation requires close monitoring. Capsule polysaccharide from all 90 pneumococcal serotypes cannot be included in one vaccine; therefore, research is focusing on alternatives, such as using common immunogenic pneumococcal proteins to be included in future vaccines.

Pneumolysin, a cytoplasmic cholesterol-dependent cytolysin, is a particularly interesting vaccine candidate because it is produced by all strains of S. pneumoniae, and is protective in animal vaccination models. Cholesterol-dependent cytolysins form large pores in cholesterol-containing membranes, and are therefore cytotoxic to mammalian cells. Pneumolysin is also known to induce inflammatory responses in the host lung similar to that caused by S. pneumoniae, activate complement in the absence of antibody, induce apoptosis, and activate Toll-like receptor 4 (TLR-4). The amino acid sequence of pneumolysin is thought to be highly conserved throughout all pneumococcal serotypes. As part of an ongoing study of pneumococcal virulence genes, the authors identified a number of clinical isolates with mutations in their pneumolysin gene (ply), predominantly in the ply gene of serotypes 1, 7, and 8. The serotype 1 isolates had additional mutations in the ply gene, and were chosen for further investigation due to their high attack rate and the recent increase in serotype 1 disease. 4 were serotype 1, 2 with mutations within the ply gene. Analysis of 28 additional serotype 1 isolates revealed that more than half had mutations within the ply gene, which resulted in the abrogation of the toxins hemolytic activity. Multilocus sequence typing (MLST) of the serotype 1 isolates revealed a correlation between mutations in the ply gene and sequence type.

Cell extracts were prepared from single colonies of cell extracts from each serotype 1 S. pneumoniae isolate, and diluted to known concentrations to give a baseline from which pneumolysin expression levels and hemolytic activity could be measured. Pneumolysin levels were evaluated by Western blotting and ELISA, and hemolytic activity using TEM. Pneumococcal cell extracts were incubated with an equal volume of 2% erythrocyte suspension, washed to remove unbound toxin, and run on SDS-PAGE for Western blotting with antipneumolysin antibody. This allowed analysis of pneumolysins ability to bind to erythrocytes. Transmission electron microscopy was used to assess the toxins ability to form pores on host cell membranes. Cell extract from two strains representing two pneumolysin alleles was prepared as described earlier, then filtered using 0.2 micron syringe filters. Equal volumes of cell extract, containing equivalent total protein levels, were mixed with a 2% erythrocyte suspension and incubated for 30 min at 37°C. The cells were lysed and membranes were washed five times with distilled water to remove unbound toxin. The membranes were then resuspended in a quarter of the total volume. Five microliters of sample was placed onto glow-discharged carbon-coated grids and negatively stained with NanoVan according to the product instructions. Grids were viewed at a magnification of 25,000 with an LEO 912 energy filter transmission electron microscope.

This resulted in the expression of nonhemolytic forms of pneumolysin. Pneumolysin from both ST306 and ST227 bound to erythrocytes. Analysis of pore formation by transmission electron microscopy revealed that pneumolysin expressed by ST306 was unable to form pores on erythrocyte membranes, in comparison with ST227 pneumolysin, Arcs were observed on membranes treated with cell extract from both STs, but the Ply 306 toxin did not assemble to form functional pores in the host cell membrane. PBS-treated membrane examined as a negative control was similar in appearance to ST306-treated membrane without the presence of arc structures. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing "wild-type" pneumolysin were ST227. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD. Although pneumolysin may be less conserved than previously assumed, antibodies raised against wild-type pneumolysin still recognized the pneumolysin expressed by all serotype 1 pneumococci in Western blots and ELISA, and therefore this protein is still a viable protein component of next generation conjugate vaccines.

A nontoxic form of pneumolysin would be a highly desirable starting point for vaccine production, since it would produce immunity against almost the complete range of S. pneumoniae. Previous pneumolysin mutants have reduced activity, but retain residual toxicity. In their second recent paper, Kirkham and co-workers describe the construction of a series of mutations in Ply, in a region implied to have a role in oligomerization. These were then evaluated for hemolytic activity using the same negative staining TEM procedure described above. The resulting mutants had no hemolytic activity, yet all mutants were recognized by monoclonal antibody Ply4, that recognizes an important antigenic region of Ply and can block oligomerization. One single-amino-acid-deletion Ply mutant, delta-A146 Ply, was demonstrated to be unable to form pores in cell membranes or stimulate the in vivo inflammatory effects associated with native Ply treatment. Mice vaccinated with delta-A146 Ply plus alum have high titers of neutralizing anti-Ply immunoglobulin G (IgG) and are protected from S. pneumoniae infection significantly longer than mice given alum alone. This mutant is nontoxic at concentrations greater than 1,000 times that of the native toxin; it was found to be as immunogenic as native pneumolysin without the associated effects such as production of the inflammatory mediators interleukin-6 and cytokine-induced neutrophil chemoattractant KC, damage to lung integrity, and hypothermia in mice. Vaccination with this mutant protects mice from challenge with S. pneumoniae. Incorporation of this mutant pneumolysin into current pneumococcal vaccines may increase their efficacy.

References:

• Kirkham, L. A.; Jefferies, J. M.; Kerr, A. R.; Jing, Y.; Clarke, S. C.; Smith, A., and Mitchell T. J.: Identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin. J. Clin. Microbiol., 44, 151-159 (2006).
  • Abstract (courtesy of the Journal of Clinical Microbiology).

• Kirkham, L. A.; Kerr, A. R.; Douce, G. R.; Paterson, G. K.; Dilts, D. A.; Liu, D. F, and Mitchell, T. J.: Construction and immunological characterization of a novel nontoxic protective pneumolysin mutant for use in future pneumococcal vaccines. Infect. Immun., 74, 586-93 (2006).
  • Abstract (courtesy of Infection and Immunity).

• Owen, R. H.; Boulnois, G. J.; Andrew, P. W., and Mitchell, T. J.: A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae. FEMS Microbiol. Lett., 121, 217221 (1994).
  • Abstract (courtesy of Science Direct).

Chow and group, in a recent Biophysical Journal article, describe the use of negative stain transmission electron microscopy with Nano-W in conjunction with light scattering to study the formation and dissociation of aggregates during the unfolding and re-folding of apomyoglobin. Biophysical characterization of nonfunctional protein aggregates at physiologically relevant temperatures is needed to better understand the kinetic and thermodynamic relationships between protein folding and misfolding. Dynamic and static laser light scattering were used for the detection and detailed characterization of apomyoglobin (apoMb) soluble aggregates populated at room temperature upon dissolving the purified protein in buffer at pH 6.0, both in the presence and absence of high concentrations of urea. Unlike the beta-sheet self-associated aggregates found previously for this protein at high temperatures, the resulting soluble aggregates have either a-helical or random coil secondary structure, depending on solvent and solution conditions. Hydrodynamic diameters range from 80 to 130 nm, and a semiflexible chain-like morphology was found. Low pH combined with high urea concentration led to structural unfolding and complete elimination of the large aggregates. Even upon starting from this virtually monomeric unfolded state, however, protein refolding led to the formation of severely self-associated species with native-like secondary structure. Under these conditions, kinetic apoMb refolding was found to proceed via two parallel routes: one leading to native monomer, and the other leading to a misfolded and heavily self-associated state bearing native-like secondary structure.

ApoMb-containing solutions were imaged using a JEOL 100CX transmission electron microscope. Samples were stained with Nano-W (methylamine tungstate) negative stain and placed on a pioloform coating grid support film before data analysis.

Reference:

Chow, C.; Kurt, N.; Murphy, R. M., and Cavagnero, S.: Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution. Biophys. J., 90, 298-309 (2006).

• Abstract (courtesy of the Biophysical Journal).

More information:

• Catalog information: Negative stain reagents
• NanoVan: Product information and instructions
• Nano-W: Product information and instructions
• Technical help: Negative staining
• Negative staining: Other references

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Inosine Monophosphate Dehydrogenase, Functionally Analyzed with Nanogold®

A prototypic uncompetitive inhibitor of IMPDH is mycophenolic acid (MPA), the active form of mycophenolate mofeteil (CellCept®), a widely used immunosuppressive drug. Ji and co-workers, in their recent paper in the Journal of Biological Chemistry, report that MPA interacts with intracellular IMPDH in vivo to alter its mobility on SDS-polyacrylamide gels. Immunofluorescence microscopy showed that it also induces a striking conformational change in IMPDH protein in intact cells, resulting in the formation of annular aggregates of protein, with concomitant inhibition of IMPDH activity. These aggregates are not associated with any known intracellular organelles and their formation may be reversed by incubating cells with guanosine, which repletes intracellular GTP, or with GTP-gamma-S. Enzymatic activity of IMPDH, measured by the increase in absorbance at 290 nm (formation of XMP) was restored by GTP also restores IMPDH activity. Treatment of highly purified IMPDH with MPA also results in the formation of large aggregates of protein; this process is both prevented and reversed by the addition of GTP. GTP also binds to IMPDH at physiologic concentrations, and was shown by immunofluorescence to induce the formation of linear arrays of tetrameric protein; furthermore, it prevents the aggregation of protein by MPA.

The composition of the large aggregates was examined in more detail by immunogold staining. For immunogold staining, cells were fixed with 1% glutaraldehyde and blocked with 1% BSA in phosphate-buffered saline for 30 minutes, then incubated with rabbit anti-IMPDH antibody (1 : 300 dilution) in 1% BSA for 1 hour. The cells were then washed, and incubated with 1 : 200 Nanogold® anti-Rabbit IgG for 1 hour at room temperature. The cells were then post-fixed with 1% glutaraldehyde. Nanogold particles were then silver enhanced using HQ Silver Enhancement kit according to the product instructions. The ultrathin sections were stained with lead citrate solution and examined using a Philips CM12 transmission electron microscope. The results confirmed that the annular and linear arrays were composed of IMPDH protein. The steps in protein oligomerization were then evaluated by electron microscopy using negative staining with uranyl acetate, which showed the formation and assembly of protein tetramers.

It was concluded that intracellular GTP acts as an antagonist to MPA by directly binding to IMPDH and reversing the conformational changes in the protein. The regulation of IMPDH by GTP levels may be important in the multitude of biologic effects of IMPDH inhibitors, which include inhibition of T and B cell activation, induction of differentiation of leukemic cell lines, apoptosis in hematopoietic and solid tumor cell lines, impairment of maturation and function of dendritic cells, and interference with glycosylation, cell adhesion, and nitric oxide synthesis. Increased intracellular GTP levels may help reverse these effects by increasing IMPDH enzymatic activity and restoring de novo guanine nucleotide biosynthesis.

Reference:

Ji, Y.; Gu, J.; Makhov, A. M.; Griffith, J. D., and Mitchell, B. S.: Regulation of the interaction of inosine monophosphate dehydrogenase with mycophenolic Acid by GTP. J. Biol. Chem., 281, 206-212 (2006).

• Abstract (courtesy of the Journal of Biological Chemistry).

More information:

• Nanogold conjugates: Catalog information
• Nanogold-antibody conjugates: Product information
• Silver enhancement: Catalog information
• HQ Silver: Product information
• Technical help: Silver enhancement
• Pre-embedding Nanogold labeling with HQ Silver enhancement: special report
• Technical help: Nanogold conjugates
• References: Nanogold conjugates

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

New Telephone Extensions

We also extend a belated welcome to Deepali Mitra, an experienced biochemical Research Associate who joins us after a career than includes spells at the State University of New York at Stony Brook, the Department of Paediatrics in the Starship Children's Hospital, University of Auckland (New Zealand), and Brookhaven National Laboratory. Ms. Mitra is helping us to develop applications of our nanoparticle staining and detection reagents for biomedical imaging.

More information:

• Nanoprobes: People (index)
• Company information
• Nanoprobes: general contact information and directions

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other Recent Publications

Reference:

Pinto, Y. Y.; Le, J. D.; Seeman, N. C.; Musier-Forsyth, K.; Taton, T. A., and Kiehl, R. A.: Sequence-encoded self-assembly of multiple-nanocomponent arrays by 2D DNA scaffolding. Nano Lett., 5, 2399-2402 (2005).

• Abstract (courtesy of PubMed (Medline)).

Not to be outdone, Gao, Zhan and Liu describe the formation of double and multi-wall silver nanotubes in their recent Langmuir paper. The silver nanotubes were templated using uniform low-molecular-mass organogel nanotubes self-assembled from an L-glutamic-acid-based bolaamphiphile, N,N-eicosanedioyl-di-L-glutamic acid (EDGA). EDGA was used to gel a water/ethanol solvent mixture, forming helical nanotubes; when these were then mixed with silver nitrate in water/ethanol, the silver(I) cations could be coordinated with both the inner and outer surfaces of the EDGA nanotubes, probably by the carboxylic acid groups. The silver cation was then reduced by photoirradiation with ultraviolet light (254 nm, 25W) to yield double-wall silver nanotubes, in which two silver layers were separated by one EDGA layer. Longer reduction times of the mixed gels and silver nitrate in the solution resulted in the formation of three-, four-, and five-wall silver nanotubes. In these multiwall silver nanotubes, each wall was separated at a distance of about 2.7 nm, which corresponds to the length of the bolaamphiphile molecule. The authors suggest that dissolved EDGA molecules and excess silver (I) cations were further assembled onto the surface of the formed double-wall silver nanotubes, and the photoreduction then caused the formation of the third-wall silver nanotubes. The multiwall silver nanotubes were further formed in a similar way. Slow reduction was found to be necessary; treatment with sodium borohydride resulted only in the formation of a black precipitate.

Reference:

Gao, P.; Zhan, C., and Liu, M.: Controlled synthesis of double- and multiwall silver nanotubes with template organogel from a bolaamphiphile. Langmuir, 22, 775-779 (2006).

• Abstract (courtesy of PubMed (Medline)).

To date, utilizing nanoparticles for nuclear targeting has frequently proved unsuccessful due to the impermeable nature of the plasma and nuclear membranes, and nanoparticle design and synthesis is a critical factor in addressing these issues. In their recent paper in Bioconjugate Chemistry, de la Fuente and Berry describe the delivery of water-soluble gold particles to the cell nucleus by conjugation with TAT peptide. Tat protein-derived peptide sequence by a straightforward and economical methodology, by coupling the Tat protein-derived peptide sequence (GRKKRRQRRR) to tiopronin-protected 2-3 nm gold particle using EDC/Sulfo-NHS coupling chemistry. The particles were subsequently tested in vitro using a human fibroblast cell line. Biocompatibility of the gold-tiopronin and gold-Tat nanoparticles were evaluated via the cell viability of hTERT-BJ1 human fibroblasts by the MTT assay: The metabolic activity and proliferation of fibroblasts, measured after 24 hours of culture, revealed no appreciable cytotoxic effects to cells at concentration as high as 5 micromolar, and values reached 80% compared to untreated controls at concentrations higher than 10 micromolar. TEM images taken after 1 h incubation with the particles showed that both gold-tiopronin and gold-Tat were taken up into the cell body: while gold-Tat particles were mainly located in the nucleus, only a few gold-tiopronin particles appeared to be taken into the cell through membrane invaginations, and accumulated in the mitochondrial surroundings; gold-tiopronin nanoparticles were not detected in the nucleus.

Reference:

de la Fuente, J. M., and Berry C. C.: Tat peptide as an efficient molecule to translocate gold nanoparticles into the cell nucleus. Bioconjug. Chem., 16, 1176-1180 (2005).

• Abstract (courtesy of PubMed (Medline)).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .