Evaluation of back scatter interferometry, a method for detecting protein binding in solution.

Back Scatter Interferometry (BSI) has been proposed to be a highly sensitive and versatile refractive index sensor usable for analytical detection of biomarker and protein interactions in solution. However the existing literature on BSI lacks a physical explanation of why protein interactions in general should contribute to the BSI signal. We have established a BSI system to investigate this subject in further detail. We contribute with a thorough analysis of the robustness of the sensor including unwanted contributions to the interferometric signal caused by temperature variation and dissolved gasses. We report a limit of the effective minimum detectability of refractive index at the 10(-7) level. Long term stability was examined by simultaneously monitoring the temperature inside the capillary revealing an average drift of 2.0 × 10(-7) per hour. Finally we show that measurements on protein A incubated with immunoglobulin G do not result in a signal that can be attributed to binding affinities as otherwise claimed in literature.

Isoelectric focusing is superior to immunofixation electrophoresis in diagnosing CNS inflammation.

BACKGROUND: A sensitive method to detect intrathecal IgG production is important in diagnosing inflammatory central nervous system (CNS) diseases, including multiple sclerosis (MS). OBJECTIVE: To compare cerebrospinal fluid (CSF) electrophoresis with isoelectric focusing (IEF), immunofixation-peroxidase electrophoresis (IFPE) and high-resolution agarose electrophoresis with protein-staining (HRAGE). METHODS: Paired serum and CSF samples from 307 consecutive patients attending a general neurology clinic were examined with IEF, IFPE and HRAGE. Clinical diagnosis was based on review of the patients' medical records after an average of 4 years. RESULTS: The sensitivity for detecting any inflammatory (autoimmune or infectious) CNS disease (52 patients) was 67% for IEF, 50% for IFPE and 29% for HRAGE. The sensitivity for detecting MS (14 patients) was 93%, 86% and 29% respectively. The sensitivity for detecting clinically isolated syndrome (eight patients) was 75%, 25% and 13% respectively. The number of oligoclonal bands in IEF was higher in inflammatory than in non-inflammatory neurological diseases or symptoms, but similar in MS and other inflammatory diseases. CONCLUSION: IEF is the method of choice in diagnosing intrathecal IgG synthesis.

Time-course study of 1,25-(OH)2-vitamin D3 induction of homologous receptor and c-myc in nontransformed and transformed C3H/10T1/2 cell clones.

1. We have used 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to investigate autoregulation of homologous receptor and the control of c-myc mRNA and protein expression in C3H/10T1/2 cells. 2. 10 nM 1,25-(OH)2D3 stimulated 1,25-(OH)2D3 receptor (VDR) synthesis in both non-transformed C3H/10T1/2 Cl 8 and in chemically transformed C3H/10T1/2 Cl 16 cells within 4 hr of treatment. Maximal induction was observed between 8 and 24 hr. 3. Two VDR mRNA transcripts, 2.7 and 4.8 kb, were present in both cell types. There were parallel changes in VDR specific mRNA levels and cellular VDR concentration in the C3H/10T1/2 Cl 8 cells indicating that the increase in receptor concentrations was dependent on de novo mRNA synthesis. 4. The increase in VDR mRNA concentration in the chemically transformed C3H/10T1/2 Cl 16 cells was maximal already at 4 hr, preceding the maximal increase in receptor concentration by 4-6 hr. 5. Analysis of c-myc mRNA levels also showed cell line specificity. 6. The c-myc mRNA level increased 2.1-fold with 10 nM 1,25-(OH)2D3 treatment in C3H/10T1/2 Cl 8 cells after 12 hr while the C3H/10T1/2 Cl 16 cells had maximal c-myc mRNA level after 1 hr. 7. The relative amount of c-myc mRNA remained higher than that of unstimulated controls the next 10-12 hr in C3H/10T1/2 Cl 16 cells. 8. The c-myc protein levels were not affected by 1,25-(OH)2D3 treatment in either cell line as detected by Western blot analysis. 9. Our data suggest that 1,25-(OH)2D3 mediated induction of VDR does not require prior c-myc protein synthesis in the C3H/10T1/2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of 1alpha,25-dihydroxyvitamin D3 receptor in cell extracts with low protein concentration.

An unacceptable loss of tritiated 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to the wall of the reaction tubes constituted an obstacle when examining C3H/10T1/2 Cl 8 cells for 1,25-(OH)2D3 receptor. The loss of tracer in low protein cell extracts could be strongly reduced by incubating the cell extracts in polyethylene tubes and in the presence of inert peptides prepared by digestion of gluten proteins. When incubated in buffer the recovery of tracer increased from 3 to 45% by using polyethylene tubes instead of sodium-glass tubes. However, the presence of a sufficient amount of inert peptides in the buffer significantly increased the recovery of tracer to 89%. This procedure improved the saturation binding analysis of the 1,25-(OH)2D3 receptor in the C3H/10T1/2 Cl 8 cells using the hydroxylapatite assay.

Serum vitamin D metabolites and calcitriol receptor concentration in parathyroid tissue in primary hyperparathyroidism.

Vitamin D metabolites in serum and calcitriol receptor concentration in parathyroid tissue were examined in 52 patients operated on for primary hyperparathyroidism. The calcitriol receptor levels were not different in parathyroid adenomas (mean 224 fmol/mg of protein, range 29-509, N = 43), normal parathyroid tissue (mean 245, range 31-690, N = 20), and primary parathyroid hyperplasia (mean 172, range 46-477, N = 9). Preoperative serum levels of calcitriol concentration correlated inversely to the calcitriol receptor in normal parathyroid tissue in patients with adenoma (r = -0.57, N = 17, p = 0.017), but no such correlation was found in the corresponding adenomas (r = 0.14, p = 0.59). In 31 patients in whom both pre- and postoperative vitamin D metabolite analyses were carried out, 23 had lower calcitriol postoperative concentrations compared to preoperative values (p = 0.012, sign test). No change was found in the other vitamin D metabolites postoperatively. By multiple regression analysis calcitriol concentration in serum was inversely correlated to the serum concentration of urea and phosphate (p = 0.003). We conclude that calcitriol may influence calcitriol receptor expression in normal parathyroid tissue, but not in adenomatous parathyroid gland. Furthermore, serum calcitriol was correlated to the renal function, and phosphate level, and in most patients the calcitriol concentration was lower after the operation.

Reorganization of the cytoskeleton and morphological changes induced by 1,25-dihydroxyvitamin D3 in C3H/10T1/2 mouse embryo fibroblasts: relation to inhibition of proliferation.

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) on cell morphology, the cytoskeleton, and fibronectin were studied in three lines of C3H/10T1/2 mouse embryo fibroblasts in which the antiproliferative effect of the hormone had previously been investigated. We showed that 1,25(OH)2D3 induced morphological changes in the nontransformed C3H/10T1/2 Cl 8 cells, which flattened and spread out markedly. Visualization of actin and tubulin by immunocytochemistry disclosed a reorganization of the microfilament and microtubular systems. 1,25(OH)2D3 also induced an increase in cell-surface-associated fibronectin. These changes were only slight in the transformed cell line C3H/10T1/2 Cl 16 and absent in the transformed C3H/10T1/2 TPA 482 cell line. These effects were correlated with the growth inhibition induced by the hormone, and this suggests a possible relationship between the 1,25(OH)2D3-induced alterations of cell shape and of the cytoskeleton and the effects of the hormone on cell proliferation.

Regulation of cell growth, c-myc mRNA, and 1,25-(OH)2 vitamin D3 receptor in C3H/10T1/2 mouse embryo fibroblasts by calcipotriol and 1,25-(OH)2 vitamin D3.

Calcipotriol is a synthetic 1,25-(OH)2D3 analogue with high affinity for the 1,25-(OH)2D3 receptor, but with a lower affinity than 1,25-(OH)2D3 for vitamin D binding protein in serum. The inhibitory action of calcipotriol and 1,25-(OH)2D3 on proliferation of C3H/10T1/2 mouse embryo fibroblasts was examined in the non-transformed cell line Cl 8 and in the two transformed, tumorigenic cell lines Cl 16 and TPA 482. Upon exposure to 10 nmol/l calcipotriol or 1,25-(OH)2D3, the proliferation of Cl 8 cell line was almost completely suppressed, whereas both hormones had no effect on the cell lines Cl 16 and TPA 482. Calcipotriol was at least as effective as 1,25-(OH)2D3 in inducing up-regulation of the 1,25-(OH)2D3 receptor. Displacement studies showed no difference between calcipotriol and 1,25-(OH)2D3 in the affinity for the receptor present in Cl 8 or Cl 16 cell extracts. Furthermore, the inhibition of cell growth in Cl 8 cells by calcipotriol was not accompanied by any consistent change in the steady-state expression of c-myc mRNA. In conclusion, calcipotriol had potent growth inhibitory effect on the non-transformed cell line similar to 1,25-(OH)2D3. In the transformed cell lines, calcipotriol did not inhibit proliferation despite potent up-regulation of the 1,25-(OH)2D3 receptor.

Effect of 1,25-(OH)2-vitamin D3 on growth, homologous receptor and c-myc regulation in C3H/10T1/2 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) receptor concentration, cell proliferation, and the steady-state level of c-myc mRNA were examined in the C3H/10T1/2 mouse embryo fibroblasts, before and after exposing the cells to 1,25-(OH)2D3. The non-transformed, logarithmically growing C3H/10T1/2 Cl 8 cells contained a high concentration of 1,25-(OH)2D3 receptor (164 fmol/mg of protein). An up-regulation of the 1,25-(OH)2D3 receptor and a potent inhibition of cell growth were observed by exposing the cells to 10 nM 1,25-(OH)2D3. The concentration of 1,25-(OH)2D3 receptor in the two chemically transformed, tumorigenic cell lines. C3H/10T1/2 Cl 16 and C3H/10T1/2 TPA 482, was 218 and 63 fmol/mg of protein, respectively. In the two transformed cell lines, 10 nM 1,25-(OH)2D3 had only negligible effect on cell growth. In the Cl 16 cells, an up-regulation of the 1,25-(OH)2D3 receptor was demonstrated, but only a weak up-regulation was found in the TPA 482 cells by the 1,25-(OH)2D3 treatment. No major changes were found in c-myc mRNA levels by the 1,25-(OH)2D3 treatment. Despite inhibition of cell growth, the steady-state level of c-myc mRNA was slightly induced (35%, mean) in the Cl 8 cells compared to control cells. In the transformed cells, no consistent change of the c-myc level was found. In contrast to earlier reports, we did not find any correlation between the 1,25-(OH)2D3 receptor and c-myc level, nor did we find any decrease of c-myc mRNA by 1,25-(OH)2D3 treatment in the C3H/10T1/2 fibroblasts.

Relationship of the 1 alpha, 25-dihydroxyvitamin D3 receptor concentration and the DNA content in renal cell carcinomas.

A role has been suggested for 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in cell proliferation and differentiation. Therefore the concentration of the 1,25-(OH)2D3 receptors and DNA-ploidy was measured in 22 renal cell carcinomas. No relation was found, the mean 1,25-(OH)2D3 receptor concentration being 8.4 fmol/mg of protein (range 2.8-15.9) in diploid tumors and 7.0 fmol/mg of protein (range 0-27.8) in DNA aneuploid tumors. The aneuploid tumors (11 out of 22) had a heterogeneous DNA content in 9 out of 11 cases. At the time of operation, no patient had metastases. In this prospective study, one out of 9 patients with DNA aneuploid tumor had died of renal cell carcinoma (observed 33-58 months, median 42 months). None of 10 patients with diploid tumors had died (observed 40-56 months, median 52 months).

1,25-Dihydroxyvitamin D3 receptor measurement in primary renal cell carcinomas and autologous normal kidney tissue.

Recently it was reported that 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited cell growth in a cell line derived from a metastasis from renal cell carcinoma. We have examined samples from 23 primary renal cell carcinomas for 1,25-(OH)2D3 receptor content, and compared it with the concentrations in autologous normal kidney tissue. Nineteen of 23 (83%) renal cell carcinomas had detectable (above 1 fmol/mg protein) 1,25-(OH)2D3 receptor levels, and 15 of 23 (65%) had levels above 5 fmol/mg protein. Mean value for the renal cell carcinomas was 8.2 fmol/mg protein (range, 0-28 fmol/mg protein), and the mean value for autologous normal kidney tissue was 23.1 fmol/mg protein (range, 6.6-53.7 fmol/mg protein). The 1,25-(OH)2D3 receptor levels in the renal cell carcinomas were significantly lower than in the autologous normal kidney tissue (P less than 0.001). The 1,25-(OH)2D3 receptor was characterized by sucrose gradient analysis and DNA-cellulose chromatography. The features found for renal cell carcinoma were similar to the 1,25-(OH)2D3 receptor in normal human tissue. No correlation of 1,25-(OH)2D3 receptor levels to clinical parameters was found. This study shows that carcinomas originating from the kidney, the major vitamin D regulating organ, usually contain the 1,25-(OH)2D3 receptor. The receptor may have a cellular function in the transformed cell.