Locomotor changes associated with functional electrical stimulation-assisted gait training in persons with incomplete spinal cord injury

New treatments such as functional electrical stimulation (FES) have been developed that allow people with spinal cord injury (SCI) the ability to stand and walk. This study investigated the use of FES-assisted gait training for two subjects with incomplete spinal cord injury in a single subject, repeated measures A-B design. Average walking velocity, cadence and stride length were determined while walking with and without FES at baseline and post-treatment evaluations. Changes in parameters were analyzed statistically and explained in biomechanical terms. FES-assisted gait training affected modifications in the gait parameters. One subject, initially a non-reciprocal walker, was able to walk overground at a faster velocity, cadence and longer stride length. Both subjects showed gains in these parameters over time. This study provided positive evidence for the use of FES-assisted gait training for these individuals with incomplete SCI. This may indicate that FES is a potentially useful rehabilitative tool as a gait aid for persons with SCI

A Type 1 collagen substrate increases PTH/PTHrP receptor MRNA expression and suppresses PTHrP mRNA expression in UMR106-06 osteoblast -like cells

We have previously shown that the response of osteoblasts to parathyroid hormone (PTH) can be influenced at the receptor level by growth on the physiological substrate, type I collagen, or by treatment with retinoic acid. We have also shown differential expression of genes when cells of the osteoblast lineage are grown on type I collagen. The aim of this study was therefore to examine the effect of retinoic acid and growth on type I collagen on PTH/PTH-related protein (PTHrP) receptor mRNA expression in the osteosarcoma osteoblast-like cell line UMR 106-06. PTH/PTHrP receptor mRNA levels, as assessed by Northern blot, of cells grown on collagen were increased up to 2-fold compared with cells on plastic and in a concentration-dependent manner with respect to collagen. An increase was seen as early as 6 h and was maintained over a 24 h period. This was not due to increased mRNA stability. Retinoic acid decreased the level of receptor mRNA on both plastic and collagen at each time but did not alter mRNA stability. For all treatments PTH/PTHrP receptor mRNA abundance, relative to glyceraldehyde-3-phosphate dehydrogenase, increased steadily over 24 h after subculture of cells. In contrast, PTHrP mRNA levels were reduced in cells on collagen, compared with plastic. PTH-stimulated cAMP levels of cells grown on collagen were increased compared with plastic at 24 h, but not earlier. Consistent with the mRNA data, retinoic acid decreased the amplitude of cAMP responses in cells on plastic and collagen. There was no evidence for changes in adenylate cyclase per se, since forskolin-induced cAMP levels did not change with either treatment. This study shows that known modulators of osteoblast maturation also affect signal transduction in these cells by regulating gene expression of the PTH/PTHrP receptor as well as the PTHrP ligand

Type 1 collagen influence on gene expression in UMR106-06 osteoblast-like cells is inhibited by genistein

We have previously shown that an exogenous type I collagen matrix can regulate expression of mRNA for parathyroid hormone (PTH)-related protein (PTHrP) and its receptor, the PTH/PTHrP receptor, in the UMR106-06 osteogenic sarcoma cell line, which is considered to be representative of a relatively mature osteoblast phenotype. Consistent with those data, we show here that growth of UMR106-06 cells on type I collagen increased PTH/PTHrP receptor-binding capacity. Analysis of the binding data showed that the number of PTH/PTHrP receptors expressed by cells cultured on collagen was at least 2-fold greater than that of cells cultured on plastic. Expression of mRNA encoding alkaline phosphatase (ALP) and osteopontin (OP) was also upregulated in cells cultured on collagen, suggesting that interaction with collagen promotes the osteoblast phenotype in this cell line. Retinoic acid (RA), which has also been shown to promote osteoblastic differentiation, synergized with type I collagen to cause super-induction of OP mRNA. In contrast, RA abolished the collagen-induced increase in ALP mRNA and PTH/PTHrP receptor mRNA. The collagen-mediated increase in the expression of OP and PTH/PTHrP receptor mRNA, but not that of ALP, was perturbed by prior covalent modification of the collagen by non-enzymatic glycation. The collagen effects did not occur via interaction with RGD amino acid domains in type I collagen, but evidence was obtained for involvement of the DGEA amino acid cell-binding domain. The mechanism by which plating of UMR106-06 cells on a type I collagen substrate affects PTH/PTHrP receptor mRNA levels was investigated. Inhibition of cytoskeletal organization using cytochalasin D, and inhibitors of protein phosphatases, protein kinase C, phospholipase C and cyclooxygenase, did not abrogate the collagen-mediated effects. In contrast, treatment of cells with the protein tyrosine kinase inhibitor genistein, but not herbimycin A, dose-dependently abolished the collagen effects on the expression of PTH/PTHrP receptor, ALP and OP mRNA. These results show that a type I collagen substrate influences the expression of osteoblast-associated genes in a cell model of mature osteoblasts and suggests that this involves, at least in part, changes in intracellular tyrosine phosphorylation

Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases.

The frk gene encoding the enzyme fructokinase (fructose 6-phosphotransferase [EC 2.7.1.4]) from Zymomonas mobilis has been isolated on a partial TaqI digest fragment of the genome and sequenced. An open reading frame of 906 bp corresponding to 302 amino acids was identified on a 3-kbp TaqI fragment. The deduced amino acid sequence corresponds to the first 20 amino acids (including an N-terminal methionine) determined by amino acid sequencing of the purified protein. The 118 bp preceding the methionine codon on this fragment does not appear to contain a promoter sequence. There was weak expression of the active enzyme in the recombinant Escherichia coli clone under control of the lac promoter on the pUC plasmid. Comparison of the amino acid sequence with that of the glucokinase enzyme (EC 2.7.1.2) from Z. mobilis reveals relatively little homology, despite the fact that fructokinase also binds glucose and has kinetic and structural properties similar to those of glucokinase. Also, there is little homology with hexose kinases that have been sequenced from other organisms. Northern (RNA) blot analysis showed that the frk transcript is 1.2 kb long. Fructokinase activity is elevated up to twofold when Z. mobilis was grown on fructose instead of glucose, and there was a parallel increase in frk mRNA levels. Differential mRNA stability was not a factor, since the half-lives of the frk transcript were 6.2 min for glucose-grown cells and 6.6 min for fructose-grown cells

Increased upstream methylation has no influence on the overexpression of the parathyroid hormone-related protein gene in squamous cell carcinoma of the lung

Humoral hypercalcaemia of malignancy (HHM) commonly results from the excessive production of a parathyroid hormone-related protein (PTHrP) by tumours. We have previously shown malignancy is associated with increased DNA methylation in the 5' region of the PTHrP gene. In a series of patients with lung carcinoma and relatively high serum calcium levels, 3 patients showed substantially increased PTHrP gene methylation while 5 patients showed no change in methylation status in this region. Patients showed marked tumour-specific expression of PTHrP through the P1 and P3 promoters with more general tumour and non-tumour expression through the P2 promoter. The lack of potential key regulatory CpG sites in the P1 promoter and the complete demethylation in the P2 and P3 promoters suggests methylation does not influence tumour-specific expression of PTHrP. Although demethylation may be a prerequisite for P2 and P3 expression, the overexpression of the PTHrP gene in cancer cells must be mediated through mechanisms other than DNA methylation. Copyright (C) 2000 Elsevier Science Ltd.</p

Switching of G-protein Usage by the Calcium-sensing Receptor Reverses Its Effect on Parathyroid Hormone-related Protein Secretion in Normal Versus Malignant Breast Cells*

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Gαi in MMECs but coupled to Gαs in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer