99mTc bone scanning agents—IV. Chemical characterization of 99mTc(Sn)-pyrophosphate complexes

Various reaction mixtures for the preparation of ⁹⁹Tc(Sn)-pyrophosphate were investigated by means of gel chromatography. All components were radioactively labeled. The most likely composition of the complexes, which also appear in “no carrier added” preparations, was determined.At pH 7 one complex is found with the composition TcPyp₂. Two complexes are found at pH 4: TcPyp and TcPyp₂. Further, at pH 7 a polymeric technetium compound is found not containing tin or pyrophosphate.     

Preparation and biodistribution of 99mTc chelate of strophanthidin—a cardiac aglycone

Cardiac glycosides are well known to exert a specific and powerful effect on myocardial tissue, and there is a possibility that this class of compound with a ^99mTc radiolabel may behave as a superior myocardial imaging agent in comparison to ²⁰¹T1 which is at present used clinically. Because of the extreme chemical complexity of cardiac glycosides a simpler aglycone, strophanthidin was selected as the pilot compound for preliminary labelling and in vivo distribution studies.Strophanthidin was converted to 19-thiosemicarbazone which nicely accomodated ^99mTc to produce a pure radiopharmaceutical of high specific activity. The distribution pattern in animal models was studied which is in accordance with the metabolic studies performed earlier with the ligand itself.     

The activation of CD99 inhibits cell-extracellular matrix adhesion by suppressing β1 integrin affinity

CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by β(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated β(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the β(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matrix adhesion by suppressing β(1) integrin affinity. [BMB reports 2012; 45(3): 159-164].     

99mTc bone scanning agents—V. Influence of experimental conditions on the labeling efficiency and gel chromatography of 99mTc(Sn)HMDP

The preparation of ^99mTc(Sn)HMDP was investigated as a function of pH, Sn(II) and ligand concentration. HMDP could be labeled efficiently from pH 2–9. The Sn(II) and the ligand concentrations had a beneficial influence.The composition of the radiopharmaceutical under various experimental conditions was studied by means of gel chromatography on Biogel P-4. Six different complexes were found. A preparation consisted of maximally three major complexes. The presence of a particular complex was mainly determined by pH and ligand concentration. The Sn(II) concentration had little influence.     

The development of ‘safer’ compounds for biocidal use

It is important that the biocides industry continues, if not accelerates its moves towards the supply of compounds with lower toxicological thresholds and greater environmental acceptability.Over the last few years a number of pesticides have been classified as harmful to the environment. These have been deleted from company product profiles which has meant that companies have been compelled to develop compounds which are ‘safer’ to use. However, it must be remembered that biocides are designed to kill biological material and the development of the non-toxic biocide is a long way from reality.The obvious but most difficult way to approach this problem, is to develop new active ingredients, however, the cost of such activities is now becoming prohibitive. This does not mean that industry will not continue the search for new biocides, on the contrary in this article such a new group is discussed. What it does mean, is that the development of new active ingredients must run concurrently along with modifications to existing biocidal products and methods of manufacture.     

Development of 99mTc(CO)₃-dendrimer-FITC for cancer imaging

Study of fluorophore and technetium labeling of poly(amido)-amine (PAMAM) generation 4 (G4) dendrimer and its evaluation as potential molecular imaging agent in both normal and melanoma-bearing mice, are described. Dendrimers were first conjugated with FITC (fluorescein isothiocyanate). Dendrimer-FITC was then incubated with the intermediate [(99m)Tc(CO)(3)(H(2)O)(3)](+) and purified by gel filtration. Biodistribution and scintigraphy images were performed administrating (99m)Tc(CO)(3)-dendrimer-FITC to normal mice (NM) or melanoma-bearing mice (MBM). Cryostat tissue sections from MBM mice were analyzed by confocal microscopy. Radiolabeling yield of dendrimer was approx. 90%. The (99m)Tc(CO)(3)-dendrimer-FITC complex was stable for at least 24h. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with high tumor uptake that allowed tumor imaging. Confocal microscopy analysis showed cytoplasmic distribution of (99m)Tc(CO)(3)-dendrimer-FITC.     

Synthesis and biological evaluation of 99mTc(CO)3(His–CB) as a tumor imaging agent

The chlorambucil l-histidine conjugate was synthesized and radiolabeled with [^99mTc(CO)₃]⁺ core to form the ^99mTc(CO)₃(His–CB) complex. The radiochemical purity of the complex was over 90%. It had good hydrophilicity and was stable at room temperature. The high initial tumor uptake with certain retention, fast clearance from background, good tumor/non-tumor ratios and satisfactory scintigraphic images highlighted the potential of ^99mTc(CO)₃(His–CB) as a tumor imaging agent.     

Purification of complement components by hydrophobic affinity chromatography on phenyl—Sepharose: Purification of human C5

Human complement components C5 and C3 were purified with 41% and 20% yields, respectively, by euglobulin precipitation, DEAE—Sephacel ion-exchange chromatography and gel filtration. Phenyl—Sepharose chromatography allowed the complete separation of C3 and C5. C3 bound loosely on the resin whereas C5 bound firmly and was eluted with 50% glycerin solution. Gel filtration on Sephacryl S-300 allowed the depletion of C4bp and H that contaminated C5 preparations. Homogeneity of C5 and C3 preparations was demonstrated by SDS—PAGE and immunochemical analysis. C5 and C3 consisted of two chains (α, 110000; β, 75000) linked by disulfide bridges.     

Isolation of the β-galactosphingolipid coniferoside using a tumor cell proteome reverse affinity protocol

New approaches are vital to the development of marine natural products (MNP) as therapeutic leads. One of the more time consuming aspects of MNP research arises in the connection between structure and function. Here, we describe an isolation protocol that adapts tumor cell proteomes as a vehicle for MNP isolation therein uniting structural and functional analysis. Application of this method to extracts of the sponge Agelas conifera led to the isolation of a unique poly-hydroxybutyrated β-galactosphingolipid, coniferoside.     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

The metabolism of α-ionylidene compounds by Cercospora rosicola

α-Ionylidene ethanol was converted to 4′hydroxy-α -ionylidene acetic acid, 1′-deoxy-ABA and ABA by Cercospora rosicola. Both the 4′-(R) and 4′-(S-epimers of 4′-hydroxy-α-ionylidene acetic acid were detected but the configuration of the 1′-position was not established. Both epimers were metabolized to 1′-deoxy-ABA and ABA. Both the cis- and trans 1′,4′diols of ABA were also converted to ABA. 1′-Deoxy-ABA was stereospecifically hydroxylated to form ABA. 1′-Hydroxy-α-ionylidene derivatives inhibited ABA production and were only oxidised to ABA in low yield. α-Ionylidene ethanol, α-ionylidene acetic acid and both epimers of 4′-hydroxy-α-ionylidene acetic acid were identified as endogenous compounds.     

Inhibition of aggregation of amyloid β42 by arginine-containing small compounds

Aggregations of proteins are in many cases associated with neurodegenerative diseases such as Alzheimer's (AD). Small compounds capable of inhibiting protein aggregation are expected to be useful for not only in the treatment of disease but also in probing the structures of aggregated proteins. In previous studies using phage display, we found that arginine-rich short peptides consisting of four or seven amino acids bound to soluble 42-residue amyloid β (Aβ42) and inhibited globulomer (37/48 kDa oligomer) formation. In the present study, we searched for arginine-containing small molecules using the SciFinder searching service and tested their inhibitory activities against Aβ42 aggregation, by sodium dodecyl sulfate (SDS)-PAGE and thioflavine T binding assay. Commercially available Arg-Arg-7-amino-4-trifluoromethylcoumarin was found to exhibit remarkable inhibitory activities to the formation of the globulomer and the fibril of Aβ42. This chimera-type tri-peptide is expected to serve as the seed molecule of a potent inhibitor of the Aβ aggregation process.     

Comparative studies of radiocitrates in oncological models—I. 99mTc citrate and 67Ga citrate uptake by EMT-6 tumors in mice

⁶⁷Ga citrate and ^99mTc citrate (Solcocitran) were injected sequentially, with an interval of 48 h, into Balb/c mice bearing transplanted EMT-6 tumors. Tissue distributions of ⁶⁷Ga and ^99mTe were measured simultaneously at intervals of 1, 3, 5 and 8 h after injection of the ^99mTc citrate (49, 51, 53 and 56 h after ⁶⁷Ga citrate). Maximal tumor:blood ratios for ^99mGa and ^99mTc were 13.8 ± 3.2 and 4.0 ± 1.0 respectively, both occurring at the final period. The maximum tumor index (T.I. = T:B x % dose/g) for ⁶⁷Ga was 71 ± 23% 56 h after injection, and for Tc was 13 ± 12% 1 h after injection. Liver, kidney and spleen had equal or higher concentrations of radioactivity than tumor for either radiotracer. The somewhat higher tumor:blood ratio for ⁶⁷Ga citrate was offset by the time required for this optimum to be reached. Alternatively, the best ^99mTc citrate tumor:blood ratios were attained within 8 h, with less liver and gut radioactivity. These data fall within the range of results from other clinical and animal model studies of ⁶⁷Ga citrate and Tc citrate. In view of the radiation dose, the inconvenience of the 48–72 h wait, and the cost of ⁶⁷Ga, and because neither radiopharmaceutical is tumor specific, ^99mTc citrate may have a place in early oncological screening. The results are discussed as part of a comprehensive review of the ^99mTc citrate literature.     

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver -glucuronidase was studied by sequential lectin affinity chromatography. -Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed byN-[¹⁴C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography.Ulex europaeus agglutinin-agarose chromatography revealed the presence of (1-3) linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin,Ricinus communis agglutinin andPhaseolus vulgaris erythroagglutinin.     

Intrinsic affinity of protein – ligand binding by differential scanning calorimetry

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.     

多发性骨髓瘤^99mTc—MIBI显像的临床价值及研究进展

多发性骨髓瘤（MM）是一种浆细胞克隆性的恶性增殖性疾病,是最常见的易累及骨骼的肿瘤。骨骼X线是MM骨病检查的金标准。但是,普通X线对MM的检测存在很多限制。目前有研究报道^99mTc-甲氧基异丁基异睛（MIBI）显像反映MM瘤细胞负荷量及活动性的灵敏性、特异性强,本文主要就^99mTc-MIBI显像对MM疾病诊断、预后判断、疗效检测及与其它影像学检查比较的研究进行综述。     

Improved immunoscintigraphy by subcutaneous injection of 99mTc or 111In labelled F(ab′)2 fragments of an anti-melanoma monoclonal antibody

Technetium-99m and/or ¹¹¹In labelled F(ab′)₂ fragments of a melanoma associated MoAb 225.28S were injected i.v. in 80 patients affected by stage I to IV malignant melanoma. Seventy five percent of metastatic lesions already documented by other methods were detected by immunoscintigraphy, which was also capable of detecting a certain number of unknown metastases. However, we observed a lower percentage of positive scans in liver, lung and skin because of the poor tumour to background ratio. In some patients, subcutaneous (s.c.) injection allowed us to visualize documented metastases undetected by i.v. administration. An equal amount of non-specific F(ab′)₂ fragments (MoAb 4C4) injected s.c. as a negative control showed no positive scans. Clinical studies and Chromatographic patterns of patient serum samples suggest that the s.c. route of administration offers, with respect to the i.v. route, the advantage of reducing vascular background and aspecific accumulation in liver, probably because of retention of possible contaminants by the lymphatic system.