cDNA cloning and sequencing of tarantula hemocyanin subunits

Summary Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.     

Polyp dropout in a solitary cold-water coral

Coral Reefs - Scleractinian corals feature both sessile and mobile stages and diverse modes of development. In some cases, development can be reversed. Examples include polyp detachment in response...     

Sequencing complete mitochondrial and plastid genomes

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing" protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing" protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.     

Investigation on the precaecal and faecal digestibility of lactulose and inulin and their influence on nutrient digestibility and microbial characteristics

This study was conducted to determine the pre-caecal and faecal digestibility of lactulose and inulin and the influence of these substances on nutrient digestibility and microbial characteristics. In metabolic trials three of six male growing pigs (German Landrace x Pietrain) were fitted with an ileo-rectal anastomosis (IRA) in end-to-end technique with preserved ileo-caeco-colic valve. The metabolic trials were conducted from day 21-63 after surgery. The remaining pigs were used as intact partners (IN) for the IRA pigs. The experimental diets, based on corn, wheat, barley and soybean meal, were supplemented with either 1.5% lactulose or 2% inulin in replacement of diatomaceous earth (control). Pre-caecal digestibility of lactulose and inulin was assessed to be 79 and 98%, respectively. faecal digestibility was determined as 100%. The supplementation of lactulose and inulin had only minor effects on the pre-caecal and faecal digestibility of nutrients. Significant differences in nutrient digestibility were obvious between IRA and IN pigs, whereas the IRA pigs showed lower digestibility values with the exception of ether extracts (EE). Bacterial population in the digesta of IRA and IN pigs were not affected by the experimental diets except the concentration of gram-negative anaerobes, which inclined when the IRA pigs received the lactulose diet. The pH of chyme was significantly lower than the pH of faeces, however the pH was unaffected by the different supplemented diets. The concentration of volatile fatty acids (VFA) in pre-caecal chyme decreased significantly when IRA pigs received the lactulose supplemented diet whereas VFA in faeces were unaffected by the supplementation. IRA pigs administered with lactulose excreted more N via the urine, but the nitrogen balance remained unaffected. From the present investigation it can be concluded that lactulose and inulin did only partly or scarcely fulfill the expectation of acting as prebiotics in pigs.     

Safety,biodistribution, pharmacokinetics,and immunogenicity of <Superscript>99m</Superscript>Tc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck

Previous studies have shown the potential of murine and chimeric anti-CD44v6 monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) of head and neck squamous cell carcinoma (HNSCC). A limitation of these MAbs, however, appeared to be their immunogenicity. Therefore, humanized monoclonal antibody BIWA 4 (bivatuzumab), with an intermediate affinity for CD44v6, was recently selected. As a prelude to RIT, we evaluated the safety, tumor-targeting potential, pharmacokinetics, and immunogenicity of technetium-99m-labeled BIWA 4 in patients undergoing operations for primary HNSCC in this study. Ten patients were treated at BIWA 4 dose levels of 25 mg (n=3), 50 mg (n=4), and 100 mg (n=3). Patients received 2 mg of 750 MBq 99mTc-BIWA 4, together with 23-, 48-, and 98-mg unlabeled BIWA 4, respectively. Radioimmunoscintigraphy (RIS) was performed within 1 h and after 21 h, and patients underwent surgery at 48 h after injection. Biodistribution of 99mTc-BIWA 4 was evaluated by radioactivity measurements in blood, bone marrow, and in biopsies of a surgical specimen obtained 48 h after injection. BIWA 4 concentration in blood was assessed by ELISA and high performance liquid chromatography and related to soluble CD44v6 levels in serum samples. The development of human anti-human antibody (HAHA) responses was determined. Administration of 99mTc-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. A mean t1/2 in plasma of 54.8 +/- 11.5 h, 76.1 +/- 21.8 h, and 68.5 +/- 21.2 h was found for the 25-, 50-, and 100-mg dose group, respectively. No complex formation of BIWA 4 with soluble CD44v6 in blood was observed. RIS showed targeting of primary tumors and lymph node metastases in 8 of 10 and 1 of 5 patients, respectively. The highest tumor uptake and tumor to nontumor ratios were observed for the 50-mg dose group. Tumor uptake was 12.9 +/- 5.9, 26.2 +/- 3.1, and 15.4 +/- 1.9% of the injected dose (ID)/kg for the 25-, 50-, and 100-mg dose group, respectively, while the tumor to bone marrow ratios for these groups were 1.7 +/- 0.5, 3.2 +/- 1.1, and 2.0 +/- 0.6, respectively. CONCLUSION: 99mTc-BIWA 4 can safely be administered to patients with HNSCC, with absence of detectable HAHA responses. The 50-mg dose level showed the highest tumor uptake and tumor to nontumor ratios. These findings support the use of BIWA 4 for RIT studies in patients with HNSCC.     

Structure of the bc1 complex from Seculamonas ecuadoriensis,a jakobid flagellate with an ancestral mitochondrial genome

In eubacteria, the respiratory bc(1) complex (complex III) consists of three or four different subunits, whereas that of mitochondria, which have descended from an alpha-proteobacterial endosymbiont, contains about seven additional subunits. To understand better how mitochondrial protein complexes evolved from their simpler bacterial predecessors, we purified complex III of Seculamonas ecuadoriensis, a member of the jakobid protists, which possess the most bacteria-like mitochondrial genomes known. The S. ecuadoriensis complex III has an apparent molecular mass of 460 kDa and exhibits antimycin-sensitive quinol:cytochrome c oxidoreductase activity. It is composed of at least eight subunits between 6 and 46 kDa in size, including two large "core" subunits and the three "respiratory" subunits. The molecular mass of the S. ecuadoriensis bc(1) complex is slightly lower than that reported for other eukaryotes, but about 2x as large as complex III in bacteria. This indicates that the departure from the small bacteria-like complex III took place at an early stage in mitochondrial evolution, prior to the divergence of jakobids. We posit that the recruitment of additional subunits in mitochondrial respiratory complexes is a consequence of the migration of originally alpha-proteobacterial genes to the nucleus.     

Interactions of trimeric purine nucleoside phosphorylases with ground state analogues--calorimetric and fluorimetric studies

Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp. purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods. Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites.