Alkaline phosphatase production by periosteal cells at various oxygen tensions in vitro

A mammalian periosteal cell culture system was developed to investigate the metabolic response of fresh calf bone periosteal cells to various oxygen tensions in vitro. Two predominant cell phenotypes were seen in the culture system. A rapidly proliferating mat of alkaline phosphatase-negative cells supported the growth of overlying clusters of alkaline phosphatase-positive cells. The appearance and subsequent population growth of the alkaline phosphatase-positive cells correlated directly with increases in enzyme activity on biochemical assay. Alkaline phosphatase production was optimal at lower oxygen tensions (5%, 9%), which approximated capillary pO2. In addition, the preconfluence oxygen environment was more critical to the final expression of the enzyme activity than the postconfluence environment. The mechanism of the environmental regulation of alkaline phosphatase gene expression at various oxygen tensions is not known. Periosteal cells were highly sensitive to oxygen tension and expressed alkaline phosphatase enzyme activity at oxygen levels approximating capillary rather than atmospheric pO2.     

Discordant repeat size and phenotype in Kennedy syndrome

Previous reports in the literature have described correlation of increasing repeat length with severity of the phenotype, in Kennedy syndrome. We describe male siblings with different repeat lengths, with lack of expression of the phenotype in the sibling with the longer repeat length. The phenotype was identical to motor neurone disease. There is variability of expression in Kennedy syndrome and repeat length even in siblings cannot be taken as a conclusive indicator of severity. CAG repeat length cannot be used to predict the natural history of Kennedy disease. The diagnosis of Kennedy syndrome should be considered in male patients presenting with atypical motor neurone disease.     

Capacitively coupled electrical stimulation of bovine growth plate chondrocytes grown in pellet form

Pellets formed from isolated bovine growth plate chondrocytes were grown in various capacitively coupled electrical fields. The signals chosen were 0, 10, 100, 250, 500, 750, 1,000, and 1,500 V peak-to-peak, 60 kHz. The effect on cell proliferation and matrix production of these different voltages was determined by [3H]thymidine and [35S]sulfate uptake, respectively, Cyclic AMP assays were done to determine if increases in either thymidine or sulfate uptake were associated with changes in cAMP levels. Significantly increased cell proliferation occurred at 500, 750, and 1,000 V peak to peak. The calculated electric fields were 1.5 to 3.0 x 10(-2) V/cm. Proliferation was significantly inhibited at 1,500 V peak-to-peak with a calculated field of 4.5 x 10(-2) V/cm. Little if any change was seen in cAMP levels at 30 or 60 min following application of the appropriate electric signals.     

In vitro bone-cell response to a capacitively coupled electrical field. The role of field strength, pulse pattern, and duty cycle.

Newborn rat calvarial bone cells were grown to confluence and subjected to a matrix of sine wave 60-kHz capacitively coupled electrical signals of various field strengths, pulse-burst patterns, and duty cycles. Both [3H] thymidine incorporation into DNA and alkaline phosphatase activity were evaluated in field strengths ranging from 0.0001 to 20 mV/cm, with pulse-burst patterns ranging from continuous to 5 milliseconds ON/495 milliseconds OFF, with daily duty cycles ranging from 0.25% to 25%. A significant increase in proliferation occurred in field strengths of 0.1, 1, and 20 mV/cm when the signal was applied continuously for six hours. Significant proliferation also occurred when the 20-mV/cm field was pulsed for six hours at 5 milliseconds ON/495 milliseconds OFF and at 5 milliseconds ON/245 milliseconds OFF. No change in alkaline phosphatase activity occurred in the 20-mV/cm field with any signal. At 1 mV/cm, there was a significant decrease in alkaline phosphatase activity in the continuous signal and in the 5 milliseconds ON/62 milliseconds OFF signal; in the lower fields evaluated, there was an actual decrease in alkaline phosphatase activity with some of the signals. The field strength plays a dominant role in determining the bone-cell's proliferative response, and to a lesser extent the alkaline phosphatase activity response, to a capacitively coupled electric field. The pulse configuration and the duty cycle are also important, but only if the proper field strength is being applied to the cell.     

Uroscopy in the 21st century: high-field NMR spectroscopy

From the experiments described, it can be seen that there are different research approaches that can be taken and these are summarized in Table 1. Whereas much scientific research is principally hypothesis led, there remains, nevertheless, an important place for exploratory research. High resolution NMR can measure, directly and simultaneously, a wide range of endogenous metabolites in biological fluids and has the unique capability of providing structural information on the metabolites detected. It has proved to be a powerful research tool with which to study inherited metabolic diseases, renal disease, drug metabolism, and toxicity, and can be used to monitor the effects of drug therapy. For instance, by using a library of experimental toxins one can map the metabolic profile of site-specific nephron injury. With this approach in man one could eventually take an unknown disease such as Balkan nephropathy and predict the initial site of tubular injury, the mode of injury and therefore the kind of toxin capable of producing that injury. NMR spectroscopic techniques are still advancing rapidly, with ever increasing sensitivity and sophistication of NMR pulse sequences to enhance structural elucidation in complex mixtures. Given the advances in directly coupled HPLC-NMR and even HPLC-NMR-mass spectroscopy it is likely that these technologies in conjunction with pattern recognition will make major contribution to our understanding of renal processes and provide new diagnostic insights in the 21st century.      

Activation of guinea pig eosinophil respiratory burst by leukotriene B4: role of protein kinase C

Leukotriene B4 (LTB4) and the protein kinase C activator, 4-beta-phorbol dibutyrate (PDBu), both induced a pronounced and concentration-dependent stimulation of hydrogen peroxide (H2O2) generation by purified guinea pig peritoneal eosinophils in the concentration range 1 nM-1 microM. The LTB4 response was inhibited competitively by the specific LTB4 receptor antagonist, U-75302, with a KB of 25 nM, while the concentration-response curves for both stimuli were shifted rightwards (3.8-fold and 2.8-fold for LTB4 and PDBu, respectively) by the competitive protein kinase C inhibitor, 1-O-hexadecyl-2-O-methylglycerol at a concentration of 300 microM. LTB4 appears, therefore, to induce respiratory burst in eosinophils via a receptor-mediated mechanism involving protein kinase C.     

Pulsing electromagnetic field stimulation of the in vitro growth plate

Specific pulsing electromagnetic fields (PEMFs) have been used to stimulate growth and repair of osteogenic tissues; however, the basis for this specificity is unknown. Previously, we determined the relevant electromagnetic field parameters of the clinically used PEMF and independently verified the beneficial effects of PEMFs on the rabbit fibula fracture healing model. The goal of the present study was to develop an in vitro model that would permit the effectiveness of various electric and magnetic field components of the PEMF to be determined. The costochondral junction (CCJ) of the 21-day-old rat was exposed in vitro to PEMFs with various electric and magnetic field component amplitudes. Response of this model to PEMFs was determined by nondestructive macrophotographic measurement of CCJ growth. Preliminary data indicated that temperature effects were present in this in vitro system. Subsequent experiments designed to separate the effects of temperature and PEMFs on the growth of CCJs in tissue culture were performed. Results indicate that accurate and frequent temperature measurements must be made for in vitro models being used to study effects of PEMFs. Small temperature differences induced by the coils used to produce PEMFs in the CCJ experimental system can have significant stimulatory effects, and the combined effects of temperature and PEMFs are not linearly additive in this model. Furthermore, our results suggest that thermal and PEMF stimuli could affect macrophotographically measured growth of the CCJ by separate mechanisms or could have a synergistic effect. Therefore, PEMF stimulation experiments should be performed under strictly "athermal" conditions.     

Localization of a gene for otosclerosis to chromosome 15q25-q26

Among white adults otosclerosis is the single most common cause of hearing impairment. Although the genetics of this disease are controversial, the majority of studies indicate autosomal dominant inheritance with reduced penetrance. We studied a large multi- generational family in which otosclerosis has been inherited in an autosomal dominant pattern. Five of16 affected persons have surgically confirmed otosclerosis; the remaining nine have a conductive hearing loss but have not undergone corrective surgery. To locate the disease- causing gene we completed genetic linkage analysis using short tandem repeat polymorphisms (STRPs) distributed over the entire genome. Multipoint linkage analysis showed that only one genomic region, on chromosome 15q, generated a lod score >2.0. Additional STRPs were typed in this area, resulting in a lod score of 3.4. STRPs FES (centromeric) and D15S657 (telomeric) flank the 14. 5 cM region that contains an otosclerosis gene.