Noninvasive assessment of the functional neovasculature in 9L-glioma growing in rat brain by dynamic 1H magnetic resonance imaging of gadolinium uptake.

Pathophysiologic parameters of the functional neovasculature and the blood-brain barrier of 9L-glioma in rat brain were measured noninvasively by dynamic 1H magnetic resonance imaging studies of gadolinium (Gd)-DTPA uptake. Changes of apparent [Gd-DTPA] uptake in time (CT[t]) were analyzed in a slice through the center of 10 9L-gliomas using fast T1 measurements. The distribution of the contrast agent was spatially correlated with the distribution of perfused microvessels as determined by immunohistochemical analysis. This method permits a distinction between perfused and nonperfused microvessels with a disrupted blood-brain barrier. In transverse slices of the whole tumor, a spatial correlation was observed between CT maps and the two-dimensional distribution of perfused microvessels. In the next step, Gd-DTPA uptake rates were spatially related to the perfused microvessel density (Np) or vascular surface area (Sp). In tumor voxels with perfused microvessels, a linear correlation was found between Gd-DTPA uptake rate constants (k values) and Np or Sp. No correlation was observed between k values and the total microvessel density. These are the first data that show a relation between Gd-DTPA uptake rates and parameters of the functional neovasculature in 9L-glioma growing in rat brain. Now that Gd-DTPA uptake studies can be related to parameters of the functional neovasculature, they may be used more efficiently as a prognostic tool before or during therapy.

Determination of in vivo rat muscle Gd-DTPA relaxivity at 6.3 T.

For the in vivo relaxivity of Gd-DTPA at 6.3 T in rat muscle a value of 2.7+/-0.5 (mM s)(-1) was found, and for the in vitro value in water 3.00+/-0.56 (mM s)(-1) at 37 degrees C. The temperature dependence of the in vitro relaxivity was -0.087 (mM s degrees C)(-1). The relation between 1/T1 and the tissue Gd-DTPA concentration is linear for the normally used in vivo Gd-DTPA concentration range.

Influence of the pharmacokinetic model on the quantification of the Gd-DTPA uptake rate in brain tumours using direct T1 measurements.

After a bolus injection of Gd-DTPA, a fast tracer uptake in rat brain tumours was already observed during the tracer bolus passage. For the quantification of the uptake rate constant, pharmacokinetic models are commonly used. For a good quantification, the changes of the plasma tracer concentration directly after the bolus injection must be incorporated into these models as prior knowledge. The aim of this paper is to investigate whether or not it is necessary to include the bolus passage into the description for the plasma tracer concentration. The result of this study indicates that the best quantification of the uptake rate constant is achieved by using only the data points after the bolus passage. Using all the data points and incorporating the bolus passage in the pharmacokinetic models results in a less accurate estimation.

NMR perfusion and metabolic imaging and some examples on brain tumours.

The potentials and limitations of proton Magnetic Resonance to map the spatial distribution of perfusion parameters and of metabolite concentrations in the brain are demonstrated and discussed. Some examples of applications to brain tumours are given.

Spatial resolution and spectral information content of proton NMR spectroscopic imaging of tumour metabolism.

The value of metabolic proton NMR spectroscopic imaging to detect and classify tumours increases with the spatial resolution and the information content of the spectra. Several factors influencing these quantities are discussed.

3D neuro-electronic interface devices for neuromuscular control: design studies and realisation steps.

In order to design the shape and dimensions of new 3D multi-microelectrode information transducers properly, i.e. adapted to the scale of information delivery to and from peripheral nerve fibres, a number of studies were, and still are, being performed on modelling and simulation of electrical volume conduction inside and outside nerves, on animal experiments on stimulation and recording with single wires and linear arrays, and on new technologies for 3D micro-fabrication. This paper presents a selection of the results of these "Neurotechnology' studies at the University of Twente. The experimental and simulation results apply primarily to the peripheral motor nerves of the rat, but are also of interest for neural interfacing with myelinated nerves in man, as fascicles in man are about the same size as in the rat.

Transcriptional activation by the E1A regions of adenovirus types 40 and 41.

In order to establish whether the poor growth of the two fastidious adenoviruses types 40 and 41 (Ad40 and Ad41) in HeLa cells is due to a reduced trans-activation by the early region 1A (E1A), we have determined the trans-activating effect of this region on the expression of the chloramphenicol acetyltransferase (CAT) gene controlled by the Ad2 E4 promoter. Cotransfection of HeLa cells with plasmids containing the E1A regions of Ad5, Ad40, and Ad41, respectively, and the CAT gene controlled by the Ad2 E4 promoter showed that activation of the E4 promoter by the E1A regions of Ad40 and Ad41 depends on the same sequence elements of the E4 promoter as activation by the Ad5 E1A gene products. The level of activation, however, is significantly lower. This might partly explain the reduced growth in HeLa cells of the two viruses.

Structure and organization of the left-terminal DNA regions of fastidious adenovirus types 40 and 41.

To determine whether differences in structure and organization of the transforming regions of Ad40 and Ad41 could explain the fastidious growth of these viruses, we have sequenced these regions and analyzed the structure of the corresponding mRNAs. These regions are 85% homologous to each other and exhibit 52% of homology to the analogous Ad5 region. Like the other adenoviruses, the Ad40 and Ad41 transforming regions contain two transcriptional units, E1a and E1b. Differences and similarities in the strategic sequences of these transcription units and Ad5 regulatory elements are discussed. Northern blotting and S1-nuclease analysis of RNA isolated from transformed cells reveal that in Ad40-transformed cells region E1a is transcribed into three partially overlapping mRNAs, while no transcription of region E1b can be detected. In Ad41-transformed cells only one E1a mRNA is found, comparable to the larger Ad40 E1a mRNA. In the Ad41 E1b region a single mRNA species is synthesized, which is comparable to the E1b messenger found in cells transformed by other adenovirus serotypes. Comparison of the large proteins encoded by the Ad40 and Ad41 E1a regions with the corresponding Ad5 E1a protein shows a relatively low homology in three conserved regions. These results are discussed in relation to the transforming capacity and the fastidious growth of Ad40 and Ad41 in conventional cell lines.

Cell transformation by the left terminal regions of the adenovirus 40 and 41 genomes.

To study the transforming capacity of the two fastidious adenoviruses 40 and 41 (Ad40 and Ad41), we have cloned the left terminal regions of their genomes. Transfection with plasmids containing these regions leads to transformation of primary baby rat kidney (BRK) cells. Cell lines derived from transformed cells show an intermediate transformed phenotype. The left terminal region was present in at least 50 copies in cells transformed by Ad41 and in 2-3 copies in Ad40-transformed cells. The integration patterns of the plasmids containing the left terminal region of Ad41 were similar in three different cell lines.

Transformation of human 143 tk- cells with plasmids containing the gene encoding the adenovirus DNA-binding protein.

Human cell lines that contain and express the gene encoding the adenovirus type 5 DNA-binding protein (Ad5 DBP) are very useful for the isolation of adenovirus mutants with an altered DBP. In order to obtain these cells, human 143 tk- cells were transfected, using the calcium phosphate technique, with plasmids containing the Ad5 DBP gene and the herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker. Characterization of several tk+ transformants revealed that these cells did contain the HSV tk gene, but in none of these cells could Ad5 DBP DNA sequences be detected. However, when 143 tk- cells were co-transfected with a plasmid containing the Ad5 DBP gene and another plasmid carrying early region E1, integration of the Ad5 DBP gene in chromosomal DNA could be detected. Integration of Ad5 DNA sequences was also observed when transfection was performed with plasmids containing the Ad5 DBP gene and the long terminal repeat of Moloney murine leukaemia virus. By employing a radioimmunoassay it could be shown that DBP-related proteins were synthesized in two of the cell lines containing the Ad5 DBP gene. Since both cell lines support the growth of the temperature-sensitive viral DBP mutant, H5ts125, at the non-permissive temperature, the DBP-related proteins expressed in these cells must be functional.

Physicochemical properties of the DNAs of the fastidious adenovirus species 40 and 41.

The DNAs of the two fastidious adenovirus species 40 and 41 (Ad40 and Ad41) have been studied by restriction endonuclease cleavage, CsCl density gradient centrifugation, and liquid-phase hybridization. For Ad40 DNA, cleavage maps were constructed for the restriction endonucleases EcoRI, SalI, ClaI, BstEII, BclII, and PvuI. The EcoRI, SalI, ClaI, and XhoI maps could be established for Ad41 DNA. The buoyant density in CsCl of DNAs of the fastidious species is 1.711 g/cm3 corresponding to a G + C content of 52%. The homology of the DNAs from Ad40 and Ad41 proved to be 62-69%. These results are consistent with previous reports on antigenic and DNA restriction enzyme analysis in which the classification into two species was proposed (J. C. De Jong, R. Wigand, A. H. Kidd, G. Wadell, J. G. Kapsenberg, C. J. Muzerie, A. G. Wermenbol, and R.-G. Fritzlaff, J. Med. Virol. 11, 215-231 (1983), and I. Uhnoo, G. Wadell, L. Svensson, and M. Johansson, Dev. Biol. Stand. 53, 311-318 (1983].

Expression of integrated viral DNA sequences outside the transforming region in eight adenovirus-transformed cell lines.

The expression of early and intermediate-early viral regions in eight adenovirus type 5 transformed cell lines was analyzed by radioimmuno-inhibition and RNA-DNA hybridization techniques. Details on the arrangement of the integrated viral DNA sequences in these cell lines have already been published (Visser, L., Wassenaar, A.D.C., Van Maarschalkerweerd, M.W. and Rozijn, T.H. (1981) J. Virol, 39, 684-693). In all cell lines tested, proteins encoded by the transforming region E1 are present. Dependent on the viral DNA content, additional early regions are expressed in most cell lines. In two of the cell lines polypeptides related to the adenoviral terminal protein, encoded by the recently described region E2b, could be detected. The viral DNA sequence encoding the body of the terminal protein mRNA is probably integrated intact, but the promoter region and at least some of the leaders are lacking in these cell lines.

Nucleotide sequence analysis of the linked left and right hand terminal regions of adenovirus type 5 DNA present in the transformed rat cell line 5RK20.

A peculiar phenomenon is observed in several adenovirus type 2 or 5 (Ad2 or Ad5) transformed cell lines: the right hand and left hand terminal regions of the viral genome present in the viral DNA insertions of these cell lines are found to be linked together. A large part of the viral DNA insertion present in the Ad5 transformed rat cell line 5RK20 has been cloned in the lambda vector Charon21A, including the segment containing the linked terminal regions. Sequence analysis of the linkage region showed a perfect homology with the Ad5 DNA sequence and a direct linkage of basepair (bp) 63 of the left hand end of the viral genome to bp 108 of the right hand end. No cellular or rearranged viral sequences were present. Our findings suggest that the joining of viral sequences into the cellular genome.

Sequence organization of a viral DNA insertion present in the adenovirus-type-5-transformed hamster line BHK268-C31.

The hamster cell line BHK268-C31 contains two large viral inserts which both include sequences from the left-hand end of adenovirus type 5 (Ad5) DNA. One of these viral inserts has been cloned in the lambda vector Charon 4A. Electron microscopic analysis and restriction enzyme mapping shows that the recombinant carries a 4.4-kb-long colinear segment of viral DNA, which is located between map positions 1.5 and 14.2 in the Ad5 genome. The junctions between viral DNA and flanking sequences have been sequenced and found not to show any specific features. One of the junctions is located in the E1a coding region, 573 bp from the left-hand end of the Ad5 genome, whereas the other junction is situated in the coding region for polypeptide IVa2. The promoter region as well as the cap site for the mRNAs from region E1a are thus missing from this insert and its role in viral transformation is unclear.

Arrangement of integrated viral DNA sequences in cells transformed by adenovirus types 2 and 5.

The organization of the viral DNA sequences in 15 adenovirus-transformed cell lines was analyzed by the Southern blotting procedure. The site of adenovirus integration in the cellular genome was found not to be unique, and the viral DNA sequences involved in integration were not confined to a specific region of the adenovirus genome. Several cell lines showed simple integration patterns that demonstrated the presence of large continuous stretches of viral DNA. In four cell lines, containing sequences from both molecular ends of the viral genome, the left- and right-hand-terminal sequences appeared to be linked to each other.

The mechanism of replication of adenovirus type 5 DNA.

The analysis of replicating Ad5 DNA has led to a model for replication involving linear intermediates. The origin of replication has been localized at the molecular right end, suggesting the presence of a unique sequence of nucleotides which can be recognized by an initiation factor. On the other hand, the study of the molecular ends has shown, so far, a high degree of symmetry in the terminal nucleotide sequences of Ad5 DNA. A more detailed study of the nucleotide sequences at the ends is necessary to solve this problem.