Microwave-supported preparation of (68)Ga bioconjugates with high specific radioactivity

The generator-produced positron-emitting (68)Ga (T(1/2) = 68 min) is of potential interest for clinical PET. (68)Ga as a metallic cation is suitable for complexation reactions with chelators, naked or conjugated, with peptides or other macromolecules. Large (68)Ga generator eluate volumes, metal traces from the generator column material, or reaction reagents, however, disturb a fast, reliable, and quantitative labeling procedure. In this paper we describe a simple technique, based on anion exchange, aiming first, to increase the (68)Ga concentration, second to purify it from competing impurities, and third to obtain a fast and quantitative (68)Ga-labeled peptide conjugate that can be applied in humans without further purification. Within 5 min one can obtain from the original 6 mL generator eluate a 200 microL (68)Ga preparation (volume reduction by a factor 30) that is suitable for direct and quantitative labeling of peptide conjugates. DOTATOC (DOTA-D-Phe(1)-Tyr(3)-octreotide, DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was used as a test tracer for comparing the labeling properties of the different (68)Ga preparations. In combination with microwave heating, peptide conjugates of 0.5-1 nmol quantities could be labeled within 10 min with the full (68)Ga activity of a generator. Further purification of the (68)Ga-labeled peptide conjugate was no longer required since the nuclide incorporation was quantitative. The specific radioactivity (with respect to the peptide) was improved by a factor approximately 100 compared to the previously applied techniques using the original generator eluate. The commercial (68)Ge/(68)Ga generator from Obninsk in combination with this system for purification and concentration with an integrated microwave-supported labeling technology resulted in a kitlike technology for (68)Ga-tracer production. The first automated prototype using this technology is being tested.     

Beta-glucosylation as a part of self-resistance mechanism in methymycin/pikromycin producing strain Streptomyces venezuelae

In our study of the biosynthesis of D-desosamine in Streptomyces venezuelae, we have cloned and sequenced the entire desosamine biosynthetic cluster. The deduced product of one of the genes, desR, in this cluster shows high sequence homology to beta-glucosidases, which catalyze the hydrolysis of the glycosidic linkages, a function not required for the biosynthesis of desosamine. Disruption of the desR gene led to the accumulation of glucosylated methymycin/neomethymycin products, all of which are biologically inactive. It is thus conceivable that methymycin/neomethymycin may be produced as inert diglycosides, and the DesR protein is responsible for transforming these antibiotics from their dormant to their active forms. This hypothesis is supported by the fact that the translated desR gene has a leader sequence characteristic of secretory proteins, allowing it to be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them. Expression of desR and biochemical characterization of the purified protein confirmed the catalytic function of this enzyme as a beta-glycosidase capable of catalyzing the hydrolysis of glucosylated methymycin/neomethymycin produced by S. venezuelae. These results provide strong evidence substantiating glycosylation/deglycosylation as a likely self-resistance mechanism of S. venezuelae. However, further experiments have suggested that such a glycosylation/deglycosylation is only a secondary self-defense mechanism in S. venezuelae, whereas modification of 23S rRNA, which is the target site for methymycin and its derivatives, by PikR1 and PikR2 is a primary self-resistance mechanism. Considering that postsynthetic glycosylation is an effective means to control the biological activity of macrolide antibiotics, the availability of macrolide glycosidases, which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be a valuable part of an overall strategy for the development of novel antibiotics using the combinatorial biosynthetic approach.     

Chemiosmotic ATP synthesis in photosynthetically inactive chromoplasts from Narcissus pseudonarcissus L. linked to a redox pathway potentially also involved in carotene desaturation

Mature chromoplasts from daffodil (Narcissus pseudonarcissus) flowers, although devoid of thylakoid structures, contain immunologically detectable alpha-subunits of ATP-synthase (H(+)-transporting ATP phosphohydrolase; EC 3.6.3.14). To show the presence of the entire functional protein complex, chromoplast membrane proteins were solubilized and reconstituted in phosphatidylcholine liposomes. The membranes were energized by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential, and the initial rate of ATP synthesis was measured with a luciferin/luciferase assay. In addition, by demonstrating NADPH-dependent ATP synthesis, we show that an NAD(P)H-dependent respiratory redox pathway in chromoplasts, previously identified as an important constituent of the carotene desaturation system, proceeds concomitant with membrane energization.     

Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem)

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion.The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.     

Mutation analysis of the coding sequence of the MECP2 gene in infantile autism

Mutations in the coding region of the methyl-CpG-binding protein 2 ( MECP2) gene cause Rett syndrome and have also been reported in a number of X-linked mental retardation syndromes. Furthermore, such mutations have recently been described in a few autistic patients. In this study, a large sample of individuals with autism was screened in order to elucidate systematically whether specific mutations in MECP2 play a role in autism. The mutation analysis of the coding sequence of the gene was performed by denaturing high-pressure liquid chromatography and direct sequencing. Taken together, 14 sequence variants were identified in 152 autistic patients from 134 German families and 50 unrelated patients from the International Molecular Genetic Study of Autism Consortium affected relative-pair sample. Eleven of these variants were excluded for having an aetiological role as they were either silent mutations, did not cosegregate with autism in the pedigrees of the patients or represented known polymorphisms. The relevance of the three remaining mutations towards the aetiology of autism could not be ruled out, although they were not localised within functional domains of MeCP2 and may be rare polymorphisms. Taking into account the large size of our sample, we conclude that mutations in the coding region of MECP2 do not play a major role in autism susceptibility. Therefore, infantile autism and Rett syndrome probably represent two distinct entities at the molecular genetic level.     

Comparison of the radiotoxicity of two alpha-particle-emitting immunoconjugates,terbium-149 and bismuth-213, directed against a tumor-specific,exon 9 deleted (d9) E-cadherin adhesion protein

We investigated the effects of the alpha-particle emitters (149)Tb and (213)Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets. The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue. Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to (149)Tb and (213)Bi ((149)Tb-d9 MAb and (213)Bi-d9 MAb). The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 x 10(4) to 6 x 10(4) antigens per cell. Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min. The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by [(3)H]thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations. (149)Tb and (213)Bi immunoconjugates affected cells in suspension similarly. Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml. Proliferation after incubation with (213)Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension. In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension. For (149)Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations. However, at high activity concentrations, (149)Tb-d9 MAb had only a small effect on pelleted cells. These in vitro studies demonstrate different effects of (149)Tb and (213)Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets. Microdosimetric simulation of single cell survival after alpha-particle irradiation modeled the experimental results with reasonable accuracy.     

Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations of the NF1 gene, is characterized by multiple neurofibromas, pigmentation anomalies, and a variety of other possible complications, including an increased risk of malignant neoplasias. Tumorigenesis in NF1 is believed to follow the two-hit hypothesis postulated for tumor-suppressor genes. Loss of heterozygosity (LOH) has been shown to occur in NF1-associated malignancies and in benign neurofibromas, but only few of the latter yielded a positive result. Here we describe a systematic approach of searching for somatic inactivation of the NF1 gene in neurofibromas. In the course of these studies, two new intragenic polymorphisms of the NF1 gene, a tetranucleotide repeat and a 21-bp duplication, could be identified. Three tumor-specific point mutations and two LOH events were detected among seven neurofibromas from four different NF1 patients. Our results suggest that small subtle mutations occur with similar frequency to that of LOH in benign neurofibromas and that somatic inactivation of the NF1 gene is a general event in these tumors. The spectrum of somatic mutations occurring in various tumors from individual NF1 patients may contribute to the understanding of variable expressivity of the NF1 phenotype.