Characterization of a T-DNA insertion mutant for the protein import receptor atToc33 from chloroplasts.

In Arabidopsis thaliana, the Toc34 receptor component of the chloroplast import machinery is encoded by two independent but highly homologous genes, atToc33 and atToc34. We have isolated a T-DNA insertion mutant of atToc33 which is characterized by a pale phenotype, due to reductions in the levels of photosynthetic pigments, and alterations in protein composition. The latter involve not only chloroplast proteins but also some cytosolic polypeptides, including 14-3-3 proteins which, among other functions, have been proposed to be cytosolic targeting factors for nucleus-encoded chloroplast proteins. Within the chloroplast, many, though not all, proteins of the photosynthetic apparatus, as well as proteins not directly involved in photosynthesis, are found in significantly reduced amounts in the mutant. However, the accumulation of other chloroplast proteins is unaffected. This suggests that the atToc33 receptor is responsible for the import of a specific subset of nucleus-encoded chloroplast proteins. Supporting evidence for this conclusion was obtained by antisense repression of the atToc34 gene in the atToc33 mutant, which results in an exacerbation of the phenotype.

Three-dimensional simulation of near-infrared diffusion in tissue: boundary condition and geometry analysis for finite-element image reconstruction.

Imaging of tissue with near-infrared spectral tomography is emerging as a practicable method to map hemoglobin concentrations within tissue. However, the accurate recovery of images by using modeling methods requires a good match between experiments and the model prediction of light transport in tissue. We illustrate the potential for a match between (i) three-dimensional (3-D) frequency-domain diffusion theory, (ii) two-dimensional diffusion theory, (iii) Monte Carlo simulations, and (iv) experimental data from tissue-simulating phantoms. Robin-type boundary conditions are imposed in the 3-D model, which can be implemented with a scalar coupling coefficient relating the flux through the surface to the diffuse fluence rate at the same location. A comparison of 3-D mesh geometries for breast imaging indicates that relative measurements are sufficiently similar when calculated on either cylindrical or female breast shapes, suggesting that accurate reconstruction may be achieved with the simpler cylindrical mesh. Simulation studies directly assess the effects from objects extending out of the image plane, with results suggesting that spherically shaped objects reconstruct at lower contrast when their diameters are less than 15-20 mm. The algorithm presented here illustrates that a 3-D forward diffusion model can be used with circular tomographic measurements to reconstruct two-dimensional images of the interior absorption coefficient.

Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates.

Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.

Optimization of pelvic heating rate distributions with electromagnetic phased arrays.

Deep heating of pelvic tumours with electromagnetic phased arrays has recently been reported to improve local tumour control when combined with radiotherapy in a randomized clinical trial despite the fact that rather modest elevations in tumour temperatures were achieved. It is reasonable to surmise that improvements in temperature elevation could lead to even better tumour response rates, motivating studies which attempt to explore the parameter space associated with heating rate delivery in the pelvis. Computational models which are based on detailed three-dimensional patient anatomy are readily available and lend themselves to this type of investigation. In this paper, volume average SAR is optimized in a predefined target volume subject to a maximum allowable volume average SAR outside this zone. Variables under study include the position of the target zone, the number and distribution of radiators and the applicator operating frequency. The results show a clear preference for increasing frequency beyond 100 MHz, which is typically applied clinically, especially as the number of antennae increases. Increasing both the number of antennae per circumferential distance around the patient, as well as the number of independently functioning antenna bands along the patient length, is important in this regard, although improvements were found to be more significant with increasing circumferential antenna density. However, there is considerable site specific variation and cases occur where lower numbers of antennae spread out over multiple longitudinal bands are more advantageous. The results presented here have been normalized relative to an optimized set of antenna array amplitudes and phases operating at 100 MHz which is a common clinical configuration. The intent is to provide some indications of avenues for improving the heating rate distributions achievable with current technology.

A novel 95-kD protein is located in a linker between cytoplasmic microtubules and basal bodies in a green flagellate and forms striated filaments in vitro.

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of approximately 650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.

A Novel Basal Apparatus Protein of 90 kD (BAp90) from the Flagellate Green Alga Spermatozopsis similis is a Component of the Proximal Plates and Identifies the d-(dexter)Surface of the Basal Body.

The flagellar basal apparatus consists of the basal bodies and associated fibrous structures, and represents the organizing center for the microtubular cytoskeleton in many flagellate protists. To identify novel proteins associated with the basal bodies, basal apparatuses from the flagellate green alga Spermatozopsis similis (Chlorophyceae) were isolated and purified. A polyclonal antibody raised against a 95kD protein band enriched in purified basal apparatuses was used to screen a cDNA library of S. similis which resulted in the isolation of a full length clone coding for a novel protein of 812 amino acids (85.3kD). Sequence analysis of this clone identified extended a-helical regions and predicted several coiled-coil forming domains interrupted by spacer segments of variable lengths. A polyclonal antibody (anti-BAp90) raised against the bacterially expressed protein recognized a 90kD band (BAp90) in SDS-PAGE of isolated basal apparatuses of S. similis. Immunogold labeling using anti-BAp90 decorated the proximal plates (two striated, triangular fibers which serve as spacers between the basal bodies in their proximal region) and parts of the d-fibers (df) which interconnect the basal bodies with the microtubular d-roots and the striated microtubule-associated fibers (SMAFs). Thus, the 90kD basal apparatus protein of S. similis represents a biochemical landmark for the lateral asymmetry of the basal body identifying its d-(dexter)surface. Cytoskeletal elements containing BAp90 or structurally related proteins may be involved in the organization and/or maintenance of the positional relationship between basal bodies and the cellular cytoskeleton, and hence cell polarity.

Purification and characterization of basal apparatuses from a flagellate green alga.

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis similis by detergent extraction and mechanical disintegration. Sucrose density centrifugation yielded highly enriched basal apparatuses as shown by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated basal apparatuses. A mass spectrometric analysis of the carboxyterminal peptides of alpha tubulin documented detyrosination and glutamylation as posttranslational modifications and showed that some 5% of the alpha tubulin carries a polyglutamyl side chain which can reach at least 17 residues in length. Monoclonal antibodies raised against the purified basal apparatuses were used to characterize novel components in the basal apparatus. A 210-kD component identified by mAB BAS (basal apparatus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted with two high molecular weight bands (approximately 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end of the basal bodies. Immunofluorescence staining of isolated cytoskeletons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardtii and Dunahella bioculata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus.