[Validation of the DTP-3 system for noninvasive spinal shape measurement by comparison with X-ray examination]. / Overení presnosti systému DTP-3 urceného pro neinvazivní vysetrení tvaru pátere prostrednictvím rtg vysetrení

PURPOSE OF THE STUDY: Repeated measurements of the spine are absolutely necessary in children and adolescents affected by spinal deformities especially during their growing-up periods. To avoid risks of tissue damage from x-ray exposure, several methods for non-invasive measurement of the spinal curvature have been developed. One of them is the DTP-3 position system allowing for a three-dimensional measurement of anatomical landmarks (spinous processes) and the calculation of curvature angles in both the frontal and sagittal planes. We were interested to know whether the DTP-3 was precise enough to determine the true spinal curvature. MATERIAL AND METHODS: To determine the precision of the DTP-3 system, we constructed a model of the spine. The model was then repeatedly investigated by both the noninvasive and x-ray methods. The distortion of x-ray images caused by the central projection mechanism was considered and included in the calculation. In addition, a group of patients with scoliosis up to 40° was evaluated by both the DTP-3 system and x-ray (the latter according to Cobb's method). RESULTS: Differences in spatial coordinates between DTP-3 and x-ray examinations reached 20.9 mm in the frontal plane and 67.3 mm in the sagittal plane without distortion correction of x-ray images. The differences decreased below 1.5 mm after image distortion correction in each plane. Distortion correction had not the same effect for angle parameters as for coordinates. Differences between the DTP-3 angle parameters and Cobb's x-ray angles were below 4.7°, both without correction and after correction. The difference between DTP-3 angle parameters and Cobb's x-ray angles was -1.8° ± 3.0° (mean ± standard deviation) when measurement was performed on the patients with scoliosis. DISCUSSION: The goal of any clinical examination is to obtain data applicable to decision-making analysis. In the case of scoliosis it is necessary to report results in terms of Cobb's angle, which is the problem for all surface-dependent methods, especially in patients with double curves. A solution may be to define the maximal difference between noninvasive and x-ray methods that could be acceptable for good clinical practice. CONCLUSIONS: In this study we report good concordance between noninvasive and x-ray examinations of a modeled spinal deformity in terms of both angle and linear measurements. The same results were obtained for angle measurements in a group of patients with scoliosis up to 40°. Based on this study and our previous data we believe that the DTP-3 system can be introduced into clinical practice.

Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells.

Among retina-specific proteins, recoverin is unique with respect to its cellular regulation in that it is found in rods, cones, some bipolar cells, and a rare population of cells in the ganglion cell layer. Recoverin is a calcium-binding protein which inhibits rhodopsin kinase from phosphorylating rhodopsin. Because cells in the inner layers of the retina do not contain rhodopsin kinase, arrestin, or other phototransduction proteins, it seems likely that recoverin has a different function in those cell types. To study this protein more fully, antibodies were generated against the entire mouse recoverin protein, as well as against peptides from the amino and from the carboxyl termini. These antibodies confirmed the localization of recoverin in vivo and clearly demonstrated, in culture, cells which were recoverin positive and rhodopsin negative. Surprisingly, two unique cell phenotypes were seen in cell culture which are not found in vivo. These cells are [rhodopsin(+), recoverin(-)] and [arrestin(+), recoverin(-)]. These phenotypes appear to represent the same population of cells and suggest that the recoverin gene can be regulated independent of genes for other phototransduction proteins. This cell culture system will be useful for investigating environments and factors which participate in the expression of the recoverin gene, and may identify regulatory features of the recoverin gene which cause it to be illicitly expressed in small-cell lung carcinomas in cancer-associated retinopathy (CAR).

Functional significance of recoverin localization in multiple retina cell types.

Knowledge of the cellular localization of recoverin in photoreceptor cells has enabled its interaction with other proteins to be postulated, tested and verified. Recoverin, a calcium sensing protein, is now thought to act by prolongation of the light state through interference with the interaction of arrestin and rhodopsin. Because of the detection of recoverin in multiple cell populations, the specificity of the cellular localization of recoverin was investigated in the retina of the mouse, rat, rabbit, chicken, frog, and chameleon and compared to that for opsin, phosducin, and arrestin. In addition to photoreceptor cell staining, the application of affinity-purified antibodies against recoverin demonstrated immunoreactive cells in the inner nuclear layer and a rare immunopositive cell in the ganglion cell layer of the mouse, rat and rabbit retina. Only photoreceptor cells were stained with recoverin antibodies in the chameleon and frog retina, whereas no cells were recoverin-positive in the chicken retina. In all six species studied, only photoreceptor cells were labelled with antibodies against opsin, phosducin or arrestin. Based on intensity of staining, two distinct populations of anti-recoverin-immunoreactive cells were distinguished in the photoreceptor cell layer of the retinas of the rat and rabbit, with the more darkly stained cells (probably cones) representing about 3% of the photoreceptor cells. The presence of recoverin in cells other than photoreceptors suggests it has an alternative or additional function and indicates the presence of multiple cell type-specific expression signals in the regulatory region of the recoverin gene.

TTF-1 regulates lung epithelial morphogenesis.

TTF-1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. In vitro, TTF-1 can activate the promoter of thyroid- and pulmonary-specific genes. We postulated that TTF-1 not only is essential for the activation of tissue-specific genes, but also may directly participate in epithelial cell morphogenesis. To test this postulate, we used an antisense oligonucleotide inhibitory strategy in an in vitro model of embryonic mouse lung branching morphogenesis. This strategy suppressed TTF-1 translation and inhibited lung branching morphogenesis. The resulting abnormal phenotype was characterized by hyperplastic and unorganized proliferation of epithelial cells in the airways. The mesenchymal compartment of the lung appeared to be unaffected. These results demonstrate, for the first time, that the expression of a homeoprotein transcriptional regulator is necessary for lung epithelial morphogenesis.

Light-dependent regulation of the transcriptional activity of the mammalian gene for arrestin.

The purpose of this investigation was to determine if the cyclic variations in the amount of arrestin mRNA that occur in the mouse retina during the light/dark cycle are controlled by light, a circadian clock, or both. In this study, an experimental paradigm was designed and used to distinguish among these molecular mechanisms. The data show that the initiation and the maintenance of an increased rate of syntheses of arrestin mRNA requires light and is not dependent on the time of day. Therefore the changes in transcriptional activity of the mouse arrestin gene are controlled by light, independent of circadian clock. The mechanism for this light-dependent regulation of gene transcription in the retina is distinct from the light-dependent increases in mRNA concentrations of early response genes which occur in the suprachiasmatic nucleus and a comparative analysis of such changes in these different regions of the CNS may lead to the identification of the molecular relationship between light- and circadian-dependent gene regulation.

Developmental appearance, species and tissue specificity of mouse 23-kDa, a retinal calcium-binding protein (recoverin).

A 23-kDa, soluble, calcium-binding photoreceptor-specific protein (23-kDa) has been shown to be identical to recoverin and the cancer-associated retinopathy protein. Recoverin has been reported to activate guanylate cyclase to increase the amount of cyclic GMP and thereby reopen cation channels within the photoreceptor cells. In this study, the 23-kDa protein was purified from bovine retinas and monospecific antibodies against it were generated in rabbits. Western analysis demonstrated 23-kDa in retinas from human, monkey, bovine, dog, rabbit, rat, mouse, frog, chameleon and iguana although it was not detected in chicken or fly retinas. No immunoreactivity was observed in any non-retinal tissues except the pineal gland. The 23-kDa protein was detected, by Western analysis, at postnatal day 5 in the mouse retina and it increased in amount in parallel with the differentiation of the photoreceptor cells in normal mice and it also decreased in parallel with their degeneration in the rd mouse. Immunocytochemical analysis of the adult mouse retina showed that 23-kDa is restricted primarily to the inner segments of the photoreceptor cells and, unlike arrestin, its localization did not shift in response to light/dark changes.

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein.

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.

Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes.

The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase.

Responses of nuclear glucose-6-phosphatase to diabetes and to hydrocortisone administered to normal and diabetic rats differ from those of the microsomal enzyme.

The effect of alloxan-diabetes, and of pharmacological doses of hydrocortisone administered to normal and diabetic rats, on carbamyl phosphate:glucose phosphotransferase and D-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) activities of isolated hepatic nuclei and microsomes were studied by assay at pH 7 in the absence and presence of deoxycholate. Hormonally related alterations both in activity levels and in the activation by the detergent (i.e. latency) of activities of the two cellular structural elements differed significantly. Most strikingly, (a) a 3--4-fold increase in the levels of activities of nuclei was seen in response either to diabetes or to hydrocortisone administered to normal rats whether or not detergent was added to preparations prior to assay; (b) the normally low degree of stimulation by detergent of activities of nuclei was unaltered in diabetes, and (c) administration of the glucocorticoid to diabetic rats decreased activity levels and increased their activation by detergent. Directly contrasting responses were noted with isolated microsomal preparations. Fundamental differences in the enzymes in these two organelle preparations are thus demonstrated. It appears that both synthetic and hydrolytic activities of this enzyme of nuclei may be manifest in the presence of requisite substrates, and that activities of this organelle may become increasingly prominent under certain hormonally perturbed conditions.