Economic costs of using tailwater recovery systems for maintaining water quality and irrigation.

Best management practices (BMPs) are conservation efforts implemented to address environmental challenges associated with agricultural production. One such BMP, a tailwater recovery (TWR) system, has a dual purpose aimed at mitigating solids and nutrient losses from agricultural landscapes and creating an additional surface water source for irrigation. This study analyzes the costs of using five TWR systems to reduce solids, nutrients, and retain water. All systems were located in the Lower Mississippi Alluvial Valley and were used to irrigate crops including rice (Oryza sativa), corn (Zea mays), and soybeans (Glycine max). Costs to reduce solids and nutrients were calculated using annual payments and revenue losses due to lost tillable area from implementation of TWR systems. Similarly, cost to save and irrigate a mega-liter (ML) of water was determined as the annual payment for TWR systems, revenue losses and measured pumping cost. The range of mean total cost to reduce solids using TWR systems was $0 to $0.77 per kg; P was $0.61 to $3315.72 per kg; and N was $0.13 to $396.44 per kg. The range of mean total cost to retain water using TWR systems was $189.73 to $628.23 per ML, compared to a range of mean cost of groundwater of $13.99 to $36.17 per ML. Compared to other BMPs, TWR systems are one of the least expensive ways to reduce solid losses but remain an expensive way to reduce nutrient losses. The costs of using TWR systems to provide an additional irrigation water source range from less expensive than common conservation practices used to improve water use efficiency to more expensive and comparable to practices such as desalination. Therefore, TWR systems may be a prohibitively more expensive BMP to retain nutrients and water on some agricultural landscapes than other solutions.

Defective Bone Repair in C57Bl6 Mice With Acute Systemic Inflammation.

BACKGROUND: Bone repair is initiated with a local inflammatory response to injury. The presence of systemic inflammation impairs bone healing and often leads to malunion, although the underlying mechanisms remain poorly defined. Our research objective was to use a mouse model of cortical bone repair to determine the effect of systemic inflammation on cells in the bone healing microenvironment. QUESTION/PURPOSES: (1) Does systemic inflammation, induced by lipopolysaccharide (LPS) administration affect the quantity and quality of regenerating bone in primary bone healing? (2) Does systemic inflammation alter vascularization and the number or activity of inflammatory cells, osteoblasts, and osteoclasts in the bone healing microenvironment? METHODS: Cortical defects were drilled in the femoral diaphysis of female and male C57BL/6 mice aged 5 to 9 months that were treated with daily systemic injections of LPS or physiologic saline as control for 7 days. Mice were euthanized at 1 week (Control, n = 7; LPS, n = 8), 2 weeks (Control, n = 7; LPS, n = 8), and 6 weeks (Control, n = 9; LPS, n = 8) after surgery. The quantity (bone volume per tissue volume [BV/TV]) and microarchitecture (trabecular separation and thickness, porosity) of bone in the defect were quantified with time using microCT. The presence or activity of vascular endothelial cells (CD34), macrophages (F4/80), osteoblasts (alkaline phosphatase [ALP]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP]) were evaluated using histochemical analyses. RESULTS: Only one of eight defects was bridged completely 6 weeks after surgery in LPS-injected mouse bones compared with seven of nine defects in the control mouse bones (odds ratio [OR], 0.04; 95% CI, 0.003-0.560; p = 0.007). The decrease in cortical bone in LPS-treated mice was reflected in reduced BV/TV (21% ± 4% vs 39% ± 10%; p < 0.01), increased trabecular separation (240 ± 36 µm vs 171 ± 29 µm; p < 0.01), decreased trabecular thickness (81 ± 18 µm vs 110 ± 22 µm; p = 0.02), and porosity (79% ± 4% vs 60% ± 10%; p < 0.01) at 6 weeks postoperative. Defective healing was accompanied by decreased CD34 (1.1 ± 0.6 vs 3.4 ± 0.9; p < 0.01), ALP (1.9 ± 0.9 vs 6.1 ± 3.2; p = 0.03), and TRAP (3.3 ± 4.7 vs 7.2 ± 4.0; p = 0.01) activity, and increased F4/80 (13 ± 2.6 vs 6.8 ± 1.7; p < 0.01) activity at 2 weeks postoperative. CONCLUSION: The results indicate that LPS-induced systemic inflammation reduced the amount and impaired the quality of bone regenerated in mouse femurs. The effects were associated with impaired revascularization, decreased bone turnover by osteoblasts and osteoclasts, and by increased catabolic activity by macrophages. CLINICAL RELEVANCE: Results from this preclinical study support clinical observations of impaired primary bone healing in patients with systemic inflammation. Based on our data, local administration of VEGF in the callus to stimulate revascularization, or transplantation of stem cells to enhance bone turnover represent potentially feasible approaches to improve outcomes in clinical practice.

Defective bone repair in mast cell deficient mice with c-Kit loss of function.

KitW-sh mice carry an inactivating mutation in the gene encoding the receptor for stem cell factor, which is expressed at high levels on the surface of haematopoietic precursor cells. The mutation results in mast cell deficiency, a variety of defects in innate immunity and poorly defined abnormalities in bone. The present study was designed to characterise healing of a cortical window defect in skeletally mature KitW-sh mice using high-resolution micro computed tomographic imaging and histological analyses. The cortical bone defect healed completely in all wild type mice but failed to heal in about half of the KitW-sh mice by 12 weeks post-operative. Defective healing was associated with premature and excessive expression of TRAP positive cells embedded in fibrous marrow but with little change in ALP activity. Immuno-histochemical analyses revealed reduced CD34 positive vascular endothelial cells and F4/80 positive macrophages at 1 and 2 weeks post-operative. Impaired bone healing in the KitW-sh mice was therefore attributed to altered catabolic activity, impaired re-vascularisation and compromised replacement of woven with compact bone.

MSC-seeded dense collagen scaffolds with a bolus dose of VEGF promote healing of large bone defects.

The functional repair of large skeletal defects remains a significant challenge to orthopaedic surgeons due to the lack of effective strategies to promote bone regeneration, particularly in the elderly. This study investigated the potential use of bone marrow derived mesenchymal stromal cells (MSC) in a dense collagen scaffold with a bolus dose of vascular endothelial growth factor (VEGF) to repair a defect in the femoral diaphysis of mice. MSC isolated from bone marrow of 4-month-old donor mice were seeded in type I collagen gels that were then compressed to form scaffolds with a fibrillar density similar to osteoid. The cells remained metabolically active in scaffolds incubated in vitro for up to 15 days and differentiated into osteoblasts that deposited calcium-phosphate mineral into the scaffold, which was quantified using micro-computed tomographic (micro-CT) imaging. When implanted in a 1 mm x 3 mm unicortical defect the MSC-loaded scaffolds were rapidly mineralised and integrated into host bone with administration of 10 ng of recombinant VEGF injected into the femoral canal at 4 days postoperative. Empty scaffolds and MSC-seeded scaffolds implanted in defects that did not receive a bolus dose of VEGF did not mineralise or integrate with native bone. The approach with MSC, hydrogels and a biologic factor already approved for human use warrants further pre-clinical investigation with a large animal model.

Relationship between cartilage and subchondral bone lesions in repetitive impact trauma-induced equine osteoarthritis.

OBJECTIVE: To correlate degenerative changes in cartilage and subchondral bone in the third carpal bone (C3) of Standardbred racehorses with naturally occurring repetitive trauma-induced osteoarthritis. DESIGN: Fifteen C3, collected from Standardbred horses postmortem, were assessed for cartilage lesions by visual inspection and divided into Control (CO), Early Osteoarthritis (EOA) and Advanced Osteoarthritis (AOA) groups. Two osteochondral cores were harvested from corresponding dorsal sites on each bone and scanned with a micro-computed tomography (CT) instrument. 2D images were assembled into 3D reconstructions that were used to quantify architectural parameters from selected regions of interest, including bone mineral density and bone volume fraction. 2D images, illustrating the most severe lesion per core, were scored for architectural appearance by blinded observers. Thin sections of paraffin-embedded decalcified cores stained with Safranin O-Fast Green, matched to the micro-CT images, were scored using a modified Mankin scoring system. RESULTS: Subchondral bone pits with deep focal areas of porosity were seen more frequently in AOA than EOA but never in CO. Articular cartilage damage was seen in association with a reduction in bone mineral and loss of bone tissue. Histological analyses revealed significant numbers of microcracks in the calcified cartilage of EOA and AOA groups and a progressive increase in the score compared with CO bones. CONCLUSION: The data reveal corresponding, progressive degenerative changes in articular cartilage and subchondral bone, including striking focal resorptive lesions, in the third carpal bone of racehorses subjected to repetitive, high impact trauma.

FGF18 augments osseointegration of intra-medullary implants in osteopenic FGFR3(-/-) mice.

Enhancement of endogenous bone regeneration is a priority for integration of joint replacement hardware with host bone for stable fixation of the prosthesis. Fibroblast Growth Factor (FGF) 18 regulates skeletal development and could therefore have applications for bone regeneration and skeletal repair. This study was designed to determine if treatment with FGF 18 would promote bone regeneration and integration of orthopedic hardware in FGF receptor 3 deficient (FGFR3(-/-)) mice, previously characterized with impaired bone formation. Rigid nylon rods coated with 200 nm of titanium were implanted bilaterally in the femora of adult FGFR3(-/-) and FGFR3(+/+) mice to mimic human orthopedic hardware. At the time of surgery, LEFT femora received an intramedullary injection of 0.5 µg FGF18 (Merck Serono) and RIGHT femora received PBS as a control. Treatment with FGF18 resulted in a significant increase in peri-implant bone formation in both FGFR3(+/+) and FGFR3(-/-) mice, with the peri-implant fibrous tissue frequently seen in FGFR3(-/-) mice being largely replaced by bone. The results of this pre-clinical study support the conjecture that FGF18 could be used in the clinical setting to promote integration of orthopedic hardware in poor quality bone.

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.

Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death.

Parathyroid hormone-related peptide (PTHrP) is a mediator of cellular growth and differentiation as well as a cause of malignancy-induced hypercalcemia. Most of the actions of PTHrP have been attributed to its interaction with a specific cell surface receptor that binds the N-terminal domain of the protein. Here we present evidence that PTHrP promotes some of its cellular effects by translocating to the nucleolus. Localization of transiently expressed PTHrP to the nucleolus was dependent on the presence of a highly basic region at the carboxyl terminus of the molecule that bears homology to nucleolar targeting sequences identified within human retroviral (human immunodeficiency virus type 1 and human T-cell leukemia virus type 1) regulatory proteins. Endogenous PTHrP also localized to the nucleolus in osseous cells in vitro and in vivo. Moreover, expression of PTHrP in chondrocytic cells (CFK2) delayed apoptosis induced by serum deprivation, and this effect depended on the presence of an intact nucleolar targeting signal. The present findings demonstrate a unique intracellular mode of PTHrP action and a novel mechanism by which this peptide growth factor may modulate programmed cell death.

Parathyroid hormone-related protein as a growth regulator of prostate carcinoma.

Parathyroid hormone-related protein (PTHrP) is produced by prostate carcinoma cells and tumors, but little is known of its role in prostate carcinogenesis. The goal of this study was to evaluate PTHrP expression in the regulation of prostate carcinoma growth using human and animal models. PTHrP expression was assessed in prostate cancer cell lines in vitro. Seven of nine cell lines produced PTHrP, and increased expression was seen during cell proliferation. The MatLyLu rat prostate carcinoma model was used to determine the effects of PTHrP overexpression on prostate tumor growth. PTHrP overexpression did not alter proliferation of the cells in vitro. However, when PTHrP-overexpressing cells were injected into rat hind limbs, primary tumor growth and tumor size were significantly enhanced as compared with control cells. To evaluate PTHrP in human prostate carcinoma patients, immunohistochemistry was performed on metastatic bone lesions. Immunolocalization of PTHrP protein was found in the cytoplasm and nucleus of cancer cells in the bone microenvironment. Because nuclear localization of PTHrP has been associated with an inhibition of apoptosis, the ability of full-length PTHrP to protect prostate cancer cells from apoptotic stimuli was examined. Cells transfected with full-length PTHrP showed significantly increased cell survival after exposure to apoptotic agents as compared with cells producing no PTHrP (plasmid control) or cells transfected with PTHrP lacking its nuclear localization signal. To determine the mechanism of action of PTHrP in prostate cancer cells, the parathyroid hormone/PTHrP receptor status of the cells was determined. These cell lines did not demonstrate parathyroid hormone/PTHrP receptor-mediated binding of iodinated PTHrP or steady-state receptor message by Northern blot analysis, but they did have a detectable receptor message by reverse transcription-PCR analysis. In summary, PTHrP is expressed in many prostate cancer cell lines in vitro and in metastatic bone lesions in vivo. PTHrP expression positively influences primary tumor size in vivo and protects cells from apoptotic stimuli. These data suggest that PTHrP plays an important role in the promotion of prostate tumor establishment and/or progression.

Bacterial profile and bone healing in rats receiving cancer therapeutic doses of bisphosphonates and corticosteroids: a pilot study.

The microbial aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains undefined. This study investigated the oral microbiota and socket healing after zoledronic acid (ZA) and dexamethasone (DX) administration. Fourteen rats assigned randomly to experimental (n=8) and control (n=6) groups were injected with ZA+DX or saline, respectively, for 3 weeks prior to and 9 weeks after the extraction of left first upper and lower molars. Whole genomic DNA probes of 38 bacterial species and five Candida species were hybridized to DNA extracted from biofilm samples on exposed bone and adjacent teeth. Only experimental rats exhibited exposed bone at euthanasia. All BRONJ-like lesions were colonized by Staphylococcus pasteuri, Streptococcus parasanguinis, and Streptococcus mitis. A significant correlation was observed between the mean proportions of species colonizing BRONJ-like lesions and the teeth of experimental rats (r=0.818, P<0.001). Significant differences were seen in several species colonizing the teeth of control rats compared to experimental rats (P<0.05). Micro-computed tomography analyses revealed higher residual bone in mandibular (P=0.001) and maxillary (P=0.108) tooth sockets of experimental rats. BRONJ-like lesions were colonized mainly by non-pathogenic bacteria. ZA+DX administered to rats at doses equivalent to those given to cancer patients resulted in changes to the oral biofilm and impaired bone healing following tooth extraction.

Circulating concentrations of parathyroid hormone-like peptide in malignancy and in hyperparathyroidism.

We have examined circulating concentrations of a parathyroid hormone-like peptide (PLP) in patients with malignancies and in patients with hyperparathyroidism. The radioimmunoassay employed reacts with synthetic amino-terminal fragments of PLP but not with parathyroid hormone. Elevated plasma PLP concentrations were observed in 50% of patients with malignancy and hypercalcemia and in 15% of normocalcemic cancer patients, mean values being higher in the former group. Detectable plasma PLP concentrations were found in 2 of 39 control subjects. In 2 patients with breast cancer plasma PLP declined concomitantly with a reduction in tumor burden. Adenocarcinoma of the breast and squamous cell carcinomas were most frequently associated with high plasma PLP levels although a variety of histologic types were represented. The presence of metastases on bone scans did not correlate with either the severity of hypercalcemia or the extent of PLP elevation. Increased concentrations of plasma PLP were also observed in 4 of 20 patients with primary hyperparathyroidism and in 5 of 16 patients with chronic renal failure and secondary hyperparathyroidism. Gel filtration analysis of immunoreactive PLP in plasma from 2 hypercalcemic breast cancer patients revealed heterogeneity, with, in each case, both large (greater than 15 kD) and small (6-7 kD) molecular weight amino-terminal moieties. The results document the presence of PLP in the circulation of patients with cancer and are consistent with a pathogenetic role for PLP in the hypercalcemia of malignancy irrespective of whether skeletal metastases have occurred. PLP may also contribute to the skeletal and/or renal manifestations of hyperparathyroid states.

The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation.

Previous work has identified the parathyroid hormone-related protein (PTHrP) nucleolar targeting signal (NTS) as both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus of a variety of cells where it is believed to participate in the regulation of cell proliferation, differentiation, and apoptotic cell death. The mechanism whereby a secreted peptide, such as PTHrP, gains access to the nuclear compartment remains a question of debate. The current work examines the possibility that exogenous PTHrP is internalized and transported to the nuclear compartment by a mechanism that is dependent on preservation of the PTHrP NTS. Transiently expressed, PTHrP(1-141) was detected at the cell surface as well as in the cytoplasmic and nuclear compartments of COS-1 cells. Deletion of the NTS, or mutation of the conserved GxKKxxK motif within the NTS, effectively prevented both cell-surface binding and nuclear/nucleolar accumulation of PTHrP(1-141). A biotinylated peptide corresponding to the PTHrP NTS (PTHrP-NTS-biotin) was internalized and translocated to the nucleus and nucleolus in a time-, temperature-, and concentration-dependent manner, whereas a peptide representing a similar bipartite NTS from Nucleolin was not. Internalization and nucleolar targeting of PTHrP-NTS-biotin were indistinguishable in CFK2 cells, which express the common PTH/PTHrP receptor, and in 27m21 cells, which do not. In addition, pretreatment with a saturating dose of synthetic PTHrP(74-113) was capable of abrogating nucleolar accumulation of the PTHrP-NTS peptide, whereas pretreatment with PTHrP(1-34) or PTHrP(67-86) was not. These observations demonstrate that binding of exogenous, full-length PTHrP to the cell surface is mediated through a conserved motif embedded in the NTS and suggest that internalization and nucleolar targeting of an NTS peptide are mediated through binding to a cell surface protein distinct from the PTH/PTHrP receptor. In total, the data support the hypothesis that secreted PTHrP(1-141) can be endocytosed and targeted to the nucleolus through a mechanism that is dependent on preservation of a core motif within the PTHrP NTS.

Tumor-induced osteomalacia: clinical and basic studies.

A patient with classic clinical and biochemical features of tumor-induced osteomalacia (hypophosphatemia, phosphaturia, and undetectable serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D]) was studied before and after resection of a benign extraskeletal chondroma from the plantar surface of the foot. Presurgical laboratory evaluation was notable for normal serum concentrations of calcium, intact parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and osteocalcin, increased serum alkaline phosphate activity, and frankly elevated urinary cyclic adenosine monophosphate (cAMP) and pyridinium cross-link excretion. Quantitative histomorphometry showed severe osteomalacia and deep erosions of the cancellous surface by active osteoclasts. After resection, serum 1,25(OH)2D normalized within 24 h, while renal tubular phosphorus reabsorption and serum phosphorus did not normalized until days 2 and 3, respectively; serum Ca declined slightly, and serum intact PTH, osteocalcin, and urinary pyridinium cross-link excretion increased dramatically. Urinary cAMP excretion declined immediately after resection and then began to increase concomitant with the increase in serum intact PTH. A second bone biopsy taken 3 months after resection demonstrated complete resolution of the osteomalacia, increased mineral apposition rate (1.09 mu/day), resorption surface (9.2%), mineralizing surface (71%), and bone formation rate (0.83 mm3/mm2/day), and marked decrease in cancellous bone volume (13.1%) and trabecular connectivity compared with first biopsy. Tumor extracts did not affect phosphate transport in renal epithelial cell lines or 1 alpha-hydroxylase activity in a myelomonocytic cell line. The patient's course suggests that the normal 1,25(OH)2D and phosphorus metabolism is due to a tumor product that may be acting via stimulation of adenylate activity. Increased bone resorption prior to surgical resection suggests that the tumor may also produce an osteoclast activator. The rise in resorption surface and pyridinium cross-link excretion, increase in serum osteocalcin and bone mineralization, normalization of osteoid width, and fall in cancellous bone volume after resection are consistent with healing of osteomalacia by rapid remodeling.

Expression of FGFR3 with the G380R achondroplasia mutation inhibits proliferation and maturation of CFK2 chondrocytic cells.

A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression.

Identification and functional characterization of adenylate cyclase-linked receptors for parathyroid hormone-like peptides on immortalized human keratinocytes.

We have identified and characterized receptors for the amino-terminal domains of PTH and PTH-like peptide (PLP) on an immortalized human keratinocyte cell line, RHEK-1. Binding of both PLP-(1-34) and PTH-(1-34) to the RHEK-1 cells was consistent with a two-site model; affinities and capacities for each site were similar for the two peptides. Both peptides also stimulated adenylate cyclase activity with an equal ED50 in this cell line. Pertussis toxin pretreatment enhanced this peptide-mediated enzyme activity, suggesting linkage of the receptor to an inhibitory guanyl nucleotide-binding protein (Gi). Adenylate cyclase activity was diminished by both homologous [PLP-(1-34)] and heterologous [epidermal growth factor (EGF)] effectors. Malignant conversion of the immortalized cells with an activated H-ras oncogene to produce the RHEK-ras cell line was associated with a reduction in binding at both PLP/PTH and EGF receptors as well as a postreceptor defect in PLP/PTH-stimulated adenylate cyclase activity. The defect in enzyme activity appeared to be due in part to a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs), but not to an increase in Gi activity. Activation of the keratinocyte amino-terminal PLP/PTH receptor resulted in a small increase in [3H]thymidine incorporation, which was associated with an increase in cell numbers. This mitogenic effect was enhanced in the presence of EGF and was markedly reduced when cells were cultured in a high extracellular calcium environment. These studies demonstrate that the amino-terminal region of PLP and PTH activates adenylate cyclase-linked receptors, which are associated with mitogenesis, in RHEK-1 cells and suggest that this cell line represents a suitable model in which to examine the actions of PLP in keratinocytes.

Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion.

In previous work we showed that the chondrodysplastic phenotype of mice homozygous for a null mutation of the PTH-related peptide (PTHrP) gene was due in part to reduced proliferation and aberrant differentiation of growth plate chondrocytes. In the present study we have extended those observations by examining chondrocytes for evidence of PTH/PTHrP receptor expression, proliferation, and programmed cell death. Receptor messenger RNA and protein were expressed in chondrocytes in the resting and proliferative zones of both wild-type and mutant mice. In normal animals, expression was abundant in the area of transition between proliferative and hypertrophic chondrocytes and absent from cells in the lower hypertrophic region. On the other hand, the hypertrophic zone in mutant mice contained nonhypertrophic chondrocytes, which exhibited characteristics of proliferating cells, including PTH/PTHrP receptor expression, [3H]thymidine incorporation, and expression of proliferating cell nuclear antigen. In contrast to the situation in normal animals, some cells adjacent to the zone of vascular invasion in mutant growth plates showed biochemical and morphological evidence of programmed cell death. In addition to these alterations in the maturation of growth plate chondrocytes, homozygous mutants demonstrated signs of aberrant differentiation of periosteal precursor cells. In some specimens, clusters of chondrocytes embedded in a cartilaginous matrix were observed between the layers of periosteal osteoblasts and the bony collar in the sterna and tibiae of mice homozygous for a null mutation of the PTHrP gene. Taken together, these observations indicate that PTHrP plays a pivotal role in the orderly progression of chondrocytes through stages of proliferation, differentiation, and programmed cell death in the epiphyseal growth plate and may also facilitate the commitment of precursors to cells of the chondrocytic or osteoblastic lineages.

Amplification of the parathyroid hormone-related peptide gene in a colonic carcinoma.

PTH-related peptide (PTHrP) is the major factor responsible for humoral hypercalcemia of malignancy. This paraneoplastic syndrome has been described in association with a number of malignancies, but rarely with carcinoma of the colon. Moreover, little is known about the molecular mechanisms that underlie PTHrP overexpression in tumors. Here we report a patient who presented with hypercalcemia 6 months after resection of a neuroendocrine colonic carcinoma (tumor I). At the time of admission, intact PTH was decreased, circulating PTHrP levels were elevated, and there was tumor recurrence (tumor II). Immunohistochemical staining of paraffin-embedded sections from tumor I did not stain for PTHrP, whereas cells from tumor II stained intensely positive. Southern blot analysis and differential PCR of genomic DNAs from tumor specimens and the patient's leukocytes demonstrated amplification of the PTHrP gene in tumor II. Moreover, staining for p53 protein was evident in tumor II, but not in tumor I, consistent with the presence of a mutant form of p53 and associated loss of tumor suppressor function in the recurrent tumor. PTHrP gene amplification was also detected in one of five other tumors associated with humoral hypercalcemia of malignancy. These findings suggest that a potential mechanism contributing to PTHrP overexpression in malignancies is gene amplification, which could arise from increased genomic instability associated with the progressive stages of neoplasia.

A comparison of the in vivo biochemical responses to exogenous parathyroid hormone-(1-34) [PTH-(1-34)] and PTH-related peptide-(1-34) in man.

PTH-related peptide (PTHrP) is one of the etiological factors associated with hypercalcemia of malignancy in humans and rodents. In both in vivo and in vitro animal systems its actions mimic those of PTH; however, its bioactivity in humans has not previously been assessed. Therefore, we compared the actions of the synthetic human (h) analogs hPTHrP-(1-34) and hPTH-(1-34) when given by iv infusion to 15 healthy subjects, aged 25 +/- 3 yr. Three 12-h test infusions were given to each subject in the order: hPTH-(1-34) at a dose of 8 pmol/kg.h, an equimolar dose (8 pmol/kg.h) of PTHrP-(1-34) (low dose), and a 10-fold higher dose (80 pmol/kg.h) of hPTHrP-(1-34) (high dose). PTH infusion resulted in significant increases from basal values in serum total ionized calcium, urinary phosphate and cAMP, and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2d3]. No significant increases from basal values in any of these variables were observed during low dose PTHrP infusion. However, a 10-fold higher dose of PTHrP significantly increased serum calcium from 2.36 +/- 0.07 to 2.63 +/- 0.16 mmol/L (P less than 0.003), ionized calcium from 1.22 +/- 0.03 to 1.39 +/- 0.09 mmol/L (P less than 0.003), urinary phosphate from 0.21 +/- 0.19 to 0.31 +/- 0.16 mmol/L glomerular filtrate (P less than 0.05), urinary cAMP from 37 +/- 18 to 53 +/- 28 nmol/L glomerular filtrate (P less than 0.01), and serum 1,25-(OH)2D3 from 29.8 +/- 12.1 to 46.0 +/- 20.3 pmol/L (P less than 0.01). For each variable these changes were statistically equivalent to the increases observed during PTH infusion. The molar concentrations of circulating immunoreactive PTH-(1-34) and PTHrP-(1-34) (at the higher dose) achieved during infusion were at a ratio of 1:3. These results suggest that the in vivo actions of synthetic hPTHrP-(1-34) are comparable to those of hPTH-(1-34), but its biological activity after infusion may be less than that of hPTH-(1-34). Moreover, the increased concentrations of serum 1,25-(OH)2D3 observed with administration of hPTHrP-(1-34) are unlike the changes seen in hypercalcemia of malignancy in which levels of this vitamin D metabolite are frequently depressed.

Comparison of the biochemical responses to human parathyroid hormone-(1-31)NH2 and hPTH-(1-34) in healthy humans.

The 1-31 fragment of human PTH [hPTH-(1-31)NH2] has been shown, like hPTH-(1-34), to have anabolic effects on the skeletons of ovariectomized rats when given intermittently, but, unlike hPTH-(1-34), it does so without affecting serum calcium concentrations and does not activate the protein kinase C second messenger pathway in some target cells. To investigate the biochemical responses to hPTH-(1-31) in humans, we have directly compared it to hPTH-(1-34) during the course of slow infusions of each. Ten healthy adults, five men and five women, aged 26+/-5 yr (range, 22-37), each received 8-h continuous infusions of 8 pmol/kg.h hPTH-(1-34) and hPTH-(1-31) given in random order at least 2 weeks apart. During the infusions there were significant increases in both plasma and urinary cAMP (P < 0.05), but there were no differences in the responses between the two peptides (P = 0.362 for plasma; P = 0.987 for urine). There were also significant phosphaturic and natriuretic responses to the two peptides, which again were not different between peptides. During the infusion of hPTH-(1-34) serum ionized calcium (Ca2+) increased from 1.21+/-0.033 to 1.29+/-0.046 mmol/L (P < 0.01), and endogenous hPTH-(1-84) decreased from 29.6+/-9 to 15.0+/-5.7 pg/mL (P < 0.01), such that there was a negative correlation between them (r2 = 0.45). However, when hPTH-(1-31) was infused, neither serum Ca2+ (1.24+/-0.03 vs. 1.25+/-0.03) nor hPTH-(1-84) (26.8+/-5 vs. 30.7+/-12 pg/mL) was affected. Circulating concentrations of 1,25-dihydroxyvitamin D3 increased from 92+/-42 to 131+/-63 pmol/L (P < 0.05) during infusion of hPTH-(1-34) and from 92+/-27 to 110+/-42 pmol/L (P = NS) during hPTH-(1-31) infusion. There was also a significant increase in the urinary measure of type I collagen degradation of aminoterminal telopeptides from 78+/-45 to 101+/-51 nmol/mmol creatinine (P < 0.05) when hPTH-(1-34) was infused, but it was not affected (68+/-30 vs. 66+/-24 nmol/mmol creatinine) by hPTH-(1-31). Therefore, hPTH-(1-31) appears to be equivalent and equipotent to hPTH-(1-34) in the release of cAMP from target tissues and the renal handling of phosphate and sodium. However, at the doses employed, it does not increase serum calcium, is a weaker stimulator of the 25-hydroxyvitamin D-1alpha-hydroxylase, and does not induce rapid bone resorption.

Expression and characterization of recombinant rat parathyroid hormone-related peptide (1-141) and an amino-terminally-truncated analogue (38-141).

We have synthesized and purified recombinant parathyroid hormone related peptide (PTHrP (1-141)) and PTHrP (38-141) using an E. coli system that requires minimal purification. The cDNAs encoding PTHrP (1-141) and PTHrP (35-141) respectively were inserted into the multiple cloning site of the pTrcHis-B bacterial expression plasmid. The PTHrP encoded sequences were thereby fused at their NH2-termini to six histidine residues within the fusion protein. The recombinant plasmids were transfected into E. coli cells and PTHrP synthesis was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C. The recombinant fusion proteins were purified by binding of the histidine residues to a nickel column followed by gradient elusion and dialysis. PTHrP (1-141) was released from its fusion protein by cyanogen bromide cleavage, whereas PTHrP (38-141) was released by enzymatic digestion with enterokinase. This rapid isolation method resulted in pure PTHrP (1-141) and (38-141) as judged by SDS-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. PTHrP (1-141) stimulated cAMP accumulation and mobilized intracellular calcium ([Ca2+]i) in UMR106 osteoblast-like cells, and stimulated phosphate transport in OK/E renal cells, whereas PTHrP (38-141) was inert in these bioassays. Availability of PTHrP and its NH2-terminally truncated analogue, which lacks the sequence necessary for its hypercalcemic actions, will enable their biological activities to be examined in greater detail.