A Rapid Method for Preparing Undecalcified Sections of Bone for Autoradiographic Investigation with Short-Lived Radionuclides

To prepare sections of undecalcified bone suitable both for autoradiography with short-lived radionuclides such as 99m-technetium (t_1/2 = 6 hr) and for normal histology, rapid processing is necessary. By modifying the routine technique of embedding in plastic, sections can be obtained within 6 hours. The most important modification concerns the temperature used for the different steps in the process. The procedure has been used to localize ^99mTc labeled methylene diphosphonate for skeletal scintigraphy.     

Synthesis and biological evaluation of novel technetium-99m labeled phenylbenzoxazole derivatives as potential imaging probes for β-amyloid plaques in brain

Two uncharged (99m)Tc-labeled phenylbenzoxazole derivatives were biologically evaluated as potential imaging probes for β-amyloid plaques. The (99m)Tc and corresponding rhenium complexes were synthesized by coupling monoamine-monoamide dithiol (MAMA) and bis(aminoethanethiol) (BAT) chelating ligand via a pentyloxy spacer to phenylbenzoxazole. The fluorescent rhenium complexes 6 and 9 selectively stainined the β-amyloid plaques on the sections of transgenic mouse, and showed high affinity for Aβ((1-42)) aggregates (K(i)=11.1 nM and 14.3 nM, respectively). Autoradiography in vitro indicated that [(99m)Tc]6 clearly labeled β-amyloid plaques on the sections of transgenic mouse. Biodistribution experiments in normal mice revealed that [(99m)Tc]6 displayed moderate initial brain uptake (0.81% ID/g at 2 min), and quickly washed out from the brain (0.25% ID/g at 60 min). The preliminary results indicate that the properties of [(99m)Tc]6 are promising, although additional refinements are needed to improve the ability to cross the blood-brain barrier.     

Immunohistochemical analysis of diethylstilbestrol-induced renal tumors in adult male Syrian hamsters: evidence for relationship to peripheral nerve sheath tumors

Estrogen-induced Syrian hamster kidney tumors (SHKT) are widely used as experimental models for the study of hormonal and renal carcinogenesis. In order to characterize the direction of differentiation of SHKT, kidney sections of diethylstilbestrol (DES)-treated hamsters (1-11 months) were analyzed by immunohistochemistry using a panel of lineage-specific markers. The first tumorous buds found in animals exposed to DES for 4-6 months exhibited prominent S100, Leu-7, and vimentin immunoreactivities. Immunopositivities for neuron-specific enolase, PGP 9.5, desmin, and glial fibrillary acidic protein were mostly detected in medium-sized and large tumors after prolonged exposure to DES (> 6 months). All neoplasms, irrespective of the size and the duration of treatment, appeared negative for cytokeratin, neurofilaments, synaptophysin, and CD99 antibodies. Western blotting confirmed to a large extent the immunohistochemical observations. The systematic analysis of serial kidney sections by confocal microscopy after double immunostaining for S100 and neurofilaments revealed that early neoplastic buds could stem from S100-positive cells associated with nerves bundles. Altogether, these observations suggest that DES-induced SHKT could be related to malignant peripheral nerve sheath tumor and originate from a yet unidentified precursor cell present in the sheath of peripheral nerves.     

Synthesis and biological evaluation of 99mTc, Re-monoamine-monoamide conjugated to 2-(4-aminophenyl)benzothiazole as potential probes for beta-amyloid plaques in the brain

The benzothiazole aniline (BTA) conjugated with monoamine-monoamide (MAMA) was synthesized and then labeled with (99m)Tc. Its corresponding rhenium analogue was synthesized, and the fluorescent staining was performed in brain sections of both Tg mouse and Alzheimer's disease (AD) patient. The fluorescent rhenium complex Re-MAMA-BTA selectively bound to the amyloid aggregates in the brain sections of both APP Tg mouse and AD patient. The analogous (99m)Tc-MAMA-BTA complex could enter the normal mouse brain with high initial uptake. These results are encouraging for further exploration of their derivatives as imaging agents for Abeta plaques in the brain.     

Binding of K99 fimbriae of enterotoxigenic Escherichia coli to pig small intestinal mucin glycopeptides

Binding of purified K99 fimbriae to cryostat sections of pig small intestine was detected. Binding sites were located in the mucus layer, but not in the submucosal connective tissue. High-Mr mucin glycopeptides from pig small intestine were found to bind to K99-fimbriated enterotoxigenic Escherichia coli, in contrast to non-fimbriated cells. Sialic acid specificity of K99 fimbriae was demonstrated by the significant reduction in binding upon desialylation of mucin glycopeptides. The binding was saturable and the dissociation constant was estimated to be 6 x 10(-7) M. Fimbriated bacteria were calculated to possess 2.3 x 10(3) binding sites per cell.     

Localization of binding sites for ANF in the rat mammary gland

The localization of ANF binding sites in the mammary gland has been performed in virgin and lactating rats using an in vitro autoradiographic procedure performed on slide-mounted frozen sections. The radioligand used was rat [125I]ANF (99-126). It was demonstrated that specific ANF binding sites were present in the secretory cells and also to a lesser extent in acinar lumen. These results suggest that ANF could play some role in the production of fluid during lactation.     

A Modified Turchini Technic for the Differential Staining of Nucleic Acids

The preparation of the 9-methyl-2,3,7-trihydroxy-6-fluorone reagent for the selective staining of both desoxyribose and ribose nucleic acids is described. With slight variations this method follows the Duckert (1937) modification of the Liebermann and Lindenbaum (1904) reaction.

The present modification of the Turchini et al. (1944) staining procedure has been used on human autonomic ganglia fixed in Bouin's fluid, rat tissues, fixed in Bouin's, Zenker's, formol and for-mol-saline fixatives and mouse liver frozen and dried. The modified Turchini method has been examined primarily for its qualitative reliability by means of the following procedure. Ribonuclease treated sections were compared with adjacent sections immersed in distilled water. In succeeding steps half of these sections were stained by the modified Turchini process and the other half by Einarsson's gallocyanin chrome alum. Evidence gleaned from this and other tests indicates that 9-methyl-2,3,7-trihydroxy-6-fluorone may be used for the selective staining of desoxyribose and ribose nucleic acids.     

An efficient method for the production of isotopically enriched cholesterol for NMR

(13)C-Cholesterol was produced with high efficiency by a genetically engineered yeast strain. The method produces ～ 1 mg of cholesterol per gram of glucose using 100 ml of culture medium. Uniform 94% enrichment where the most abundant product is the fully enriched isotopomer (u-(13)C(27)) is obtained using (u-(13)C(6), 99%) glucose medium. High enrichment is very important for relaxation experiments, but for NMR applications where carbon-carbon couplings are measured, this is problematic. A good compromise between sensitivity and cost consists in diluting (u-(13)C(6), 25%) with natural-abundance glucose. With a 2:3 ratio, the maximal amount of singlets can be obtained in 1 dimensional (D) carbon and 2D heteronuclear single-quantum correlation (HSQC) spectra with 6× intensity increase relative to natural-abundance samples. The use of (1-(13)C(1)-glucose, 99%) or (2-(13)C(1)-glucose, 99%) as isotope sources allows the labeling of the cholesterol in multiple mostly nonvicinal positions and reach 45× intensity increase. As an alternative, the dilution of (u-(13)C(6), 99%) glucose can be used to simultaneously enrich eleven pairs of (13)C up to ～ 1,000× natural-abundance probability, which should be very beneficial to double-quantum NMR experiments including the INADEQUATE and related pulse sequences. The flexibility of the method and the potential to adapt the culture protocol to specific needs should find many applications in chemistry and biology and in different fields of NMR and MS.     

Submicroscopic study of bacteria and spores under the effect of peracetic acid and various aspects of the mechanism of its action]

Microbial cells (E. coli and staphylococcus) and B. cereus spores were used in this work. Peracetic acid in a concentration of 0.005% caused the death of 99% of the microbes in 20 minutes, and 0.1% peracetic acid caused the death of 99% of the spores in 45 minutes. Changes in the surface and internal structures of the cells were revealed on the ultrathin sections of microbial cells and spores after the action of the preparation. The noted changes were explained by disturbance of permeability barrier and a high reactogenic capacity of the activated oxygen of peracetic acid.     

Synthesis of stable hydrazones of a hydrazinonicotinyl-modified peptide for the preparation of 99mTc-labeled radiopharmaceuticals.

Hydrazones of a 6-hydrazinonicotinyl-modified cyclic peptide IIb/IIIa receptor antagonist were prepared in order to protect the hydrazine moiety from reaction with trace aldehyde and ketone impurities encountered during the process of manufacturing and compounding lyophilized kits used in radiolabeling with (99m)Tc. Hydrazones were prepared by either a direct reaction of the 6-hydrazinonicotinyl-modified cyclic peptide with carbonyl compounds or by conjugation of the cyclic peptide with hydrazones of succinimidyl 6-hydrazinonicotinate. Stability of the hydrazones was evaluated by treatment with formaldehyde. Hydrazones derived from simple aliphatic aldehydes underwent an exchange reaction with formaldehyde, while hydrazones of aromatic aldehydes and ketones provided the greatest level of stability when challenged with formaldehyde. We have been successful in protecting 6-hydrazinonicotinyl-modified cyclic peptides from reacting with formaldehyde, while still allowing sufficient reactivity for radiolabeling with (99m)Tc. The hydrazones of succinimidyl 6-hydrazinonicotinate are convenient and general reagents for forming 6-hydrazinonicotinyl conjugates with amino-functionalized bioactive molecules.     

Blood Serum as an Adhesive for Paraffin Sections

Diluted human and other mammalian blood serum (15 ml. of fresh blood serum diluted with 10 ml. of freshly distilled water, with 6 ml. of 5% formalin solution in distilled water added) can be used as a good adhesive for paraffin sections. It is preferable to Mayer's egg albumin-glycerol mixture because it is easily obtainable, can be quickly prepared, and sections are less subject to loosening after its use.     

Phosphine-containing HYNIC derivatives as potential bifunctional chelators for (99m)Tc-labeling of small biomolecules

Two prototype phosphine-containing HYNIC chelators, HYNIC-Kp-DPPB and HYNIC-Ko-DPPB (HYNIC = 6-hydrazinonicotinamide; K = lysine; and DPPB = diphenylphosphine-benzoic acid), have been synthesized and characterized by NMR ((1)H, (13)C, and (31)P) and LC-MS. Macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], were prepared by reacting the phosphine-containing HYNIC chelator with (99m)TcO(4)(-) in the presence of excess tricine and stannous chloride. Results from this study clearly demonstrated that both HYNIC-Kp-DPPB and HYNIC-Ko-DPPB are able to form highly stable macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], when tricine is used as the coligand. Radio-HPLC data suggest that the complex [(99m)Tc(HYNIC-Kp-DPPB)(tricine)] exists as only one detectable isomer in solution while the complex [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] has three isomers. It was also found that three isomers of [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] interconvert at elevated temperatures, suggesting that the presence of these isomers might be due conformational changes in the macrocyclic Tc chelate. The LC-MS data for both macrocyclic (99m)Tc complexes are completely consistent with the proposed composition. The phosphine-containing HYNIC chelators described in this study may have the potential as bifunctional chelators for (99m)Tc labeling of small biomolecules.     

Binding sites of atrial natriuretic peptide in tree shrew adrenal gland

Adrenal gland binding sites for atrial natriuretic peptide-(99-126) (ANP) were quantitated in tree shrew (Tupaia belangeri) by incubation of adrenal sections with (3-[125I]-iodotyrosyl28) atrial natriuretic peptide-(99-126), followed by autoradiography with computerized microdensitometry. In the adrenal glands, there are three types of ANP binding sites. One is located in the zona glomerulosa (BMax 84 +/- 6 fmol/mg protein; Kd 122 +/- 9 pM); the second in the zona fasciculata and reticularis (BMax 29 +/- 2 fmol/mg protein; Kd 153 +/- 6 pM) and the third in the adrenal medulla (BMax 179 +/- 1 fmol/mg protein; Kd 70 +/- 2 pM). Besides the influence of ANP on the regulation of adrenocortical mineralcorticoid and glucocorticoid secretion our findings raise the possibility for a local site of action of atrial natriuretic peptide in the regulation of adrenomedullary catecholamines in the tree shrew, primates and man.     

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.     

A novel ternary ligand system useful for preparation of cationic (99m)Tc-diazenido complexes and (99m)Tc-labeling of small biomolecules

This report describes a novel ternary ligand system composed of a phenylhydrazine, a crown ether-containing dithiocarbamate (DTC), and a PNP-type bisphosphine (PNP). The combination of three different ligands with (99m)Tc results in cationic (99m)Tc-diazenido complexes, [(99m)Tc(NNAr)(DTC)(PNP)]+, with potential radiopharmaceuticals for heart imaging. Synthesis of cationic (99m)Tc-diazenido complexes can be accomplished in two steps. For example, the reaction of phenylhydrazine with (99m)TcO4- at 100 degrees C in the presence of excess stannous chloride and 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (PDTA) results in the [(99m)Tc(NNPh)(PDTA)n] intermediate, which then reacts with sodium N-(dithiocarbamato)-2-aminomethyl-15-Crown-5 (L4) and N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]ethoxyethylamine (PNP6) at 100 degrees C for 15 min to give the complex, [(99m)Tc(NNPh)(L4)(PNP6)]+ in high yield (>90%). Cationic complexes [(99m)Tc(NNPh)(DTC)(PNP)]+ are stable for > or = 6 h. Their composition was determined to be 1:1:1:1 for Tc:NNPh:DTC:PNP using the mixed-ligand experiments on the tracer ((99m)Tc) level and was further confirmed by the ESI-MS spectral data of a model compound [Re(NNPh)(L4)(L6)]+. It was found that both DTCs and bisphosphines have a significant impact on the lipophilicity of their cationic (99m)Tc-diazenido complexes. Results from a (99m)Tc-labeling efficiency experiment showed that 4-hydrazinobenzoic acid (HYBA) might be useful as a bifunctional coupling agent for (99m)Tc-labeling of small biomolecules. However, the (99m)Tc-labeling efficiency of HYBA is much lower than that of 6-hydrazinonicotinic acid (HYNIC) with tricine and trisodium triphenylphosphine-3,3',3'-trisulfonate (TPPTS) as coligands.     

Synthesis and in vitro evaluation of [18F](R)-FEPAQ: a potential PET ligand for VEGFR2

Synthesis and in vitro evaluation of [(18)F](R)-N-(4-bromo-2-fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-3-yl)methoxy)-6-methoxyquinazolin-4-amine ((R)-[(18)F]FEPAQ or [(18)F]1), a potential imaging agent for the VEGFR2, using phosphor image autoradiography are described. Synthesis of 2, the desfluoroethyl precursor for (R)-FEPAQ was achieved from t-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3) in five steps and in 50% yield. [(18)F]1 was synthesized by reaction of sodium salt of compound 2 with [(18)F]fluoroethyl tosylate in DMSO. The yield of [(18)F]1 was 20% (EOS based on [(18)F]F(-)) with >99% radiochemical purity and specific activity of 1-2 Ci/μmol (n=10). The total synthesis time was 75 min. The radiotracer selectively labeled VEGFR2 in slide-mounted sections of human brain and higher binding was found in surgically removed human glioblastoma sections as demonstrated by in vitro phosphor imager studies. These findings suggest [(18)F]1 may be a promising radiotracer for imaging VEGFR2 in brain using PET.     

99mTc- and Re-labeled 6-dialkylamino-2-naphthylethylidene derivatives as imaging probes for β-amyloid plaques

Based on the conjugate strategy, two neutral ^99mTc labeled 2-(1-(6-(dialkylamino)naphthalen-2-yl)ethylidene)malononitrile (DDNP) and 1-(6-(dialkylamino)naphthalen-2-yl)ethanone (ENE) derivatives, and their corresponding rhenium complexes were synthesized. In vitro fluorescent staining indicated that the corresponding rhenium derivatives selectively stained the β-amyloid (Aβ) plaques in the brain sections of AD model mice with low background. Compared with FDDNP and FENE, the affinities of the corresponding rhenium derivatives to Aβ aggregates decreased about 10-14-fold. In vivo biodistribution experiments in normal mice showed that ^99mTc-MAMA-ENE displayed medium initial brain uptake (0.65 %ID/g at 2 min) with a reasonable washout from the brain (0.19 %ID/g at 2 h) while ^99mTc-MAMA-DDNP showed a low brain uptake (0.28 %ID/g at 2 min). Further optimize these ^99mTc-labeled tracers in order to improve their binding affinities to Aβ plaques and diffusion through the blood brain barrier may generate useful imaging agents for SPECT.     

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Common evolutionary origin for the unstable virulence plasmid pMUM found in geographically diverse strains of Mycobacterium ulcerans

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.     

Combined S99/RB51 antigen for complement fixation test for serological diagnosis of brucellosis in cattle and sheep

AIMS: To assess the efficiency of a single antigen for the complement fixation (CF) test, prepared by combining Brucella abortus smooth strain 99 (S99) with Brucella abortus rough strain RB51(RB51), in detecting cattle and sheep infected or vaccinated with Brucella spp. METHODS AND RESULTS: Serum samples from B. abortus-infected and RB51-vaccinated cattle were tested by the CF test using S99, RB51 and the combined S99/RB51 as antigens. Likewise, serum samples from Brucella melitensis-infected, RB51-vaccinated and Brucella ovis-infected sheep were tested by the CF test using S99, RB51, hot saline (HS) and combined S99/RB51 as antigens. Comparative analysis of the CF results showed that no reduction of sensitivity or specificity occurs when S99/RB51 antigen is used instead of specific antigens used separately. CONCLUSIONS: The results of this study indicated that combined S99/RB51 antigen used in the CF test, because of its specificity and sensitivity, could be used in animal brucellosis surveillance systems to improve the efficiency of the preliminary screening of herds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes an improved antigen for the CF test for the epidemiological survey of animal brucellosis. It could represent advantages over standard protocols because of its ability to detect antibody responses following infection or vaccination withBrucella strains of rough and smooth phenotype.