When utilizing streptavidin systems with Western blots of chondrocyte, osteoblast and osteoclast lysates, proteins of the molecular weights 116 kDa, 75 kDa and 67 kDa were observed to be bound by streptavidin alone. Streptavidin binding could not be blocked by pre-incubation with an RGD containing peptide. The same proteins were bound by ExtrAvidin which lacks the RGD sequence present in streptavidin. Pre-incubation with free biotin completely abolished the binding of both streptavidin and ExtrAvidin. The three proteins observed are believed to be the biotin containing carboxylases: pyruvate carboxylase, 3-methylcrotonyl carboxylase, and propionyl carboxylase. The findings of this study underscore the need to apply vigorous controls to distinguish between endogenous biotinylated proteins and biotin used as a means to amplify avidin detection systems since a wide variety of proteins with relevance to bone and cartilage biology have molecular weights similar to the biotin carboxylases.

Animals↗

PubMed1998

Relationship between TISS and ICU cost.

OBJECTIVE: To determine whether the therapeutic intervention scoring system (TISS) reliably reflects the cost of the overall intensive care unit (ICU) population, subgroups of that population and individual ICU patients. DESIGN: Prospective analysis of individual patient costs and comparison with TISS. SETTING: Adult, 12 bedded general medical and surgical ICU in a university teaching hospital. SUBJECTS: Two hundred fifty-seven consecutive patients including 52 coronary care (CCU), 99 cardiac surgery (CS) and 106 general ICU (GIC) cases admitted to the ICU during a 12-week period in 1994. A total of 916 TISS-scored patient days were analysed MAIN OUTCOME MEASURES: A variable cost (VC) that included consumables and service usage (nursing, physiotherapy, radiology and pathology staff costs) for individual patients was measured daily. Nursing costs were calculated in proportion to a daily nursing dependency score. A fixed cost (FC) was calculated for each patient to include medical, technical and clerical salary costs, capital equipment depreciation, equipment and central hospital costs. The correlation between cost and TISS was analysed using regression analysis. RESULTS: For the whole group (n = 257) the average daily FC was pound sterling 255 and daily VC was pound sterling 541 (SEM 10); range pound sterling 23-pound sterling 2,806. In the patient subgroups average daily cost (FC + VC) for CCU was pound sterling 476 (SEM 17.5), for CS pound sterling 766 (SEM 13.8) and for GIC pound sterling 873 (SEM 13.6). In the group as a whole, a strong correlation was demonstrated between VC and the TISS for each patient day (r = 0.87, p < 0.001) and this improved further when the total TISS score was compared with the total VC of the entire patient episode (r = 0.93, p < 0.001). This correlation was maintained in CCU, CS and GIC patient cohorts with only a small median difference between actual and predicted cost (2.2 % for GIC patients). However, in the individual patient, the range of error was up to +/- 65 % of the true variable cost. For the whole group the variable cost per TISS point was pound sterling 25. CONCLUSION: These results demonstrate that TISS reliably measures overall ICU population costs as well as those of the subgroups CCU, CS and GIC. However, the relationship between TISS and cost is less reliable for the individual patient.

Adolescent↗

PubMed1998

Development of avian tibial dyschondroplasia: gene expression and protein synthesis.

Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion-which was significantly enlarged-was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy.

Animals↗

PubMed1998

Type X collagen biosynthesis and expression in avian tibial dyschondroplasia.

OBJECTIVE: Tibial dyschondroplasia (TD) is an abnormality of growth plate cartilage characterized by the presence of non-vascularized, non-mineralized tissue. The objective of this study was to examine structural and functional alterations of the growth plate-specific type X collagen in TD cartilage. DESIGN: Collagen biosynthesis was examined in organ cultures and in cultured chondrocytes from normal growth plate and TD cartilage. Thermal stability of type X collagen extracted from normal and TD cartilage organ cultures to protease digestions by trypsin plus chymotrypsin or bacterial collagenase was determined. The expression of collagen genes was examined in cultured normal and TD chondrocytes. RESULTS: Synthesis of total collagen and of type X collagen was greater than threefold higher in organ cultures from the TD lesion compared with normal growth plate. The increase in type X collagen synthesis in the lesion was compensated by a reduction in the relative proportions of types II and XI collagens. The thermal denaturation and collagenase cleavage properties of purified types II and X collagens from TD cartilage were normal. The expression of type X collagen gene was threefold higher in cultured TD chondrocytes compared to chondrocytes from normal growth plate. Normal growth plate chondrocytes in primary cultures synthesized predominantly type X collagen (80% of total collagen). In contrast, TD chondrocytes synthesized mainly types I and II collagens and type X collagen represented only 22% of total collagen. TD cells initiated the synthesis of type I collagen within 5 days of primary culture, whereas normal chondrocytes did not synthesize this collagen during the same culture period. Although type X collagen synthesis was reduced in TD chondrocytes, the mRNA levels for type X collagen were substantially higher than in normal chondrocytes. CONCLUSION: Accumulation of type X collagen in TD cartilage results from its increased biosynthesis which is due largely to increased expression of the gene for this collagen, although, the chondrocyte culture studies suggest the possibility of postranscriptional defect in type X collagen synthesis or processing in TD lesion. Moreover, the TD chondrocytes in contrast with normal chondrocytes display evidence of prompt loss of their specific phenotype during short-term primary cultures.

Animals↗

PubMed1998

Effects of hypoxia on energy state and pH in resting pulmonary and femoral arterial smooth muscles.

To determine the effects of hypoxia on energy state and intracellular pH (pHi) in resting pulmonary and systemic arterial smooth muscles, we used 31P nuclear magnetic resonance spectroscopy and colorimetric and enzymatic assays to measure pHi; intracellular concentrations of ATP, phosphocreatine, creatine, and Pi; and phosphorylation potential in superfused tissue segments from porcine proximal intrapulmonary and superficial femoral arteries. Under baseline conditions (PO2 467 +/- 12.1 mmHg), energy state and total creatine (phosphocreatine + creatine) concentration were lower and pHi was higher in pulmonary arteries. During hypoxia (PO2 23 +/- 2.4 mmHg), energy state deteriorated more in femoral arteries than in pulmonary arteries. pHi fell in both tissues but was always more alkaline in pulmonary arteries. Reoxygenation reversed the changes induced by hypoxia. These results suggest that production and/or elimination of ATP and H+ was different in resting pulmonary and systemic arterial smooth muscles under baseline and hypoxic conditions. Because energy state and pHi affect a wide variety of cellular processes, including signal transduction, contractile protein interaction, and activities of ion pumps and channels, further investigation is indicated to determine whether these differences have functional significance.

Adenosine Triphosphate↗

PubMed1998

Membrane potential-dependent and -independent vasodilation in small pulmonary arteries from chronically hypoxic rats.

Chronic hypoxia is associated with altered pulmonary vasoreactivity, and it has been suggested that an increased response to voltage-dependent vasodilators may relate to enhanced Ca++ entry via voltage-dependent channels, secondary to depolarization. Few studies have been performed on small pulmonary arteries, and it is unknown whether they are depolarized after chronic hypoxia. We examined the resting membrane potential, and the actions of voltage-dependent (verapamil, levcromakalim) and -independent (isoproterenol, forskolin, papaverine) vasodilators in small ( approximately 300 microm internal diameter) pulmonary arteries from chronically hypoxic rats. The resting membrane potential was more positive in arteries after chronic hypoxia (control: -60 +/- 0.5 mV; hypoxic: -54.4 +/- 1.1 mV; P < .01), and this was reflected by a shift to the left of the response curves for K+ and 4-aminopyridine. In arteries constricted with prostaglandin F2alpha the response to verapamil and levcromakalim was increased after chronic hypoxia, although maximum prostaglandin F2alpha-induced tension was unchanged, which implies a reduction in voltage-independent constrictor mechanisms. Although vasorelaxation to isoproterenol was depressed in arteries from hypoxic rats, forskolin-induced relaxation was enhanced substantially, and because the response to the phosphodiesterase inhibitor papaverine was unchanged, we suggest that this reflects an up-regulation of adenylate cyclase. In conclusion, chronic hypoxia resulted in a significant depolarization in small pulmonary arteries, but this may explain only partly the increased efficacy of voltage-dependent vasodilators. Whether the reduction in voltage-independent constrictor mechanisms is related to the apparent up-regulation of adenylate cyclase remains to be elucidated.

Animals↗

PubMed1997

Immunolocalization of basic fibroblast growth factor in porcine epiphyseal growth plate.

Recent evidence clearly indicates that basic fibroblast growth factor (bFGF) signal transduction is essential for normal skeletal development. Here, we report that bFGF and its receptor are specifically localized in the terminal hypertrophic chondrocytes of the porcine epiphyseal growth plate, the tissue responsible for longitudinal bone growth. Similar observations were obtained with the chondrocytes immediately adjacent to resorbing blood vessels in the secondary center of ossification of the epiphysis. These results are consistent with a recent proposal that bFGF functions in coupling osteogenesis with chondrogenesis by attracting the vascular invasion of cartilage from adjacent trabecular bone.

Animals↗

PubMed1997

Evidence of increased cholecalciferol requirement in chicks with tibial dyschondroplasia.

A series of experiments was conducted to test the hypothesis that vitamin D utilization may not be as efficient in chicks with tibial dyschondroplasia (TD). The basal diet contained 1.0% Ca and 0.45% available P with no supplemental cholecalciferol (D3). Chicks from low TD (LTD) and high TD (HTD) selected lines were fed diets supplemented with various levels of vitamin D compounds and examined for rickets and TD. When chicks were fed a D3-deficient diet containing only 1.25 micrograms/kg added D3, HTD chicks had a greater incidence of severe rickets than LTD chicks (P < 0.05). The LTD chicks did not exhibit TD when fed a diet containing adequate (20 micrograms/kg) D3. The LTD chicks fed a diet supplemented with 5 micrograms/kg D3, however, had 22% incidence of TD. When HTD chicks were fed diets supplemented with 5 micrograms/kg D3 [control diet that meets NRC (1994) requirement for D3], 20 micrograms/kg D3, 5 micrograms/kg 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] or the combination of both D3 (20 micrograms/kg) and 1,25-(OH)2D3 (5 micrograms/ kg), TD incidence was highest in HTD chicks fed the control diet. When HTD chicks were fed diets with an increased dietary level of 1,25-(OH)2D3 (10 micrograms/kg) further reduction of TD incidence (P < 0.05) occurred. A potentially toxic level (Soares et al., 1983) of 1,25-(OH)2D3 (15 micrograms/kg) fed to HTD chicks resulted in still greater suppression of incidence of TD even though growth and feed intake in HTD chicks was greater than those of LTD chicks. It is concluded that the development of TD in HTD chicks is associated with subnormal ability to metabolize vitamin D.

Animals↗

PubMed1997

Vasopressin and oxytocin release during prolonged environmental hypoxia in the rat.

BACKGROUND: The mechanism causing peripheral oedema in hypoxaemic chronic obstructive pulmonary disease has not been established. Vasopressin, a powerful antidiuretic hormone involved in salt and water homeostasis, is released in response to acute hypoxia. However, the effect of prolonged hypoxaemia on hypothalamic and pituitary release of the magnocellular hypothalamic hormones, vasopressin and oxytocin, has not previously been studied. METHODS: Male Wistar rats were randomly allocated to either normobaric, hypoxic (10% O2) or control (21% O2) environmental chambers. An initial series of experiments examined plasma vasopressin concentration, osmolality, sodium concentration, packed cell volume (PCV), and weight gain at weekly intervals (n = 4-6) for six weeks. The maximum increase in plasma vasopressin concentration and PCV occurred after five weeks. In a second experiment vasopressin and oxytocin concentrations in the hypothalamus, pituitary gland, and plasma were measured in eight control and eight hypoxic rats after five weeks in the environmental chambers. RESULTS: In rats exposed to environmental hypoxia PCV increased (p < 0.001) and weight gain decreased (p < 0.05) compared with controls. The plasma vasopressin concentration increased progressively from a baseline of 1.36 (0.2) pmol/l (n = 6) to a maximum of 4.38 (0.8) pmol/l (n = 6; p < 0.01) during the first five weeks of environmental hypoxia (difference 3.02 (95% CI 1.18 to 4.86)). Plasma osmolality and sodium concentration were unchanged in hypoxic rats compared with controls during the six week period. The hypothalamic vasopressin concentration was increased (p < 0.001) after five weeks of environmental hypoxia (91.6 (4.8) pmol/ hypothalamus) compared with controls (57.4 (5.1) pmol/hypothalamus), the difference being 34.2 pmol/hypothalamus (95% CI 21.6 to 46.5). The pituitary vasopressin concentration was unchanged. In hypoxic rats hypothalamic oxytocin (59.6 (3.2) pmol/hypothalamus) was greater (p < 0.01) than in controls (42 (3.8) pmol/hypothalamus), a difference of 17.6 pmol/ hypothalamus (95% CI 8.7 to 26.5). Similarly, the plasma oxytocin concentration was increased (p < 0.05) in hypoxic rats (6.78 (1.2) pmol/l) compared with controls (3.3 (0.8) pmol/l), a difference of 3.48 pmol/l (95% CI 0.89 to 6.07). The pituitary oxytocin concentration was unchanged in the two groups. CONCLUSIONS: These results demonstrate an increase in hypothalamic production of vasopressin and oxytocin in rats during prolonged hypoxaemia. Increased plasma concentrations of neurohypophysial hormones would be expected to impair sodium and water homeostasis in patients with hypoxaemia. However, the absence of change in the plasma osmolality and sodium concentrations in this study and previous clinical investigations suggests that compensatory mechanisms modulate the actions of both vasopressin and oxytocin. A reduction in renal blood flow or decreased renal responsiveness to the neurohypophyseal hormones may be involved.

Animals↗

PubMed1997

Chondrocytes of the tibial dyschondroplastic lesion are apoptotic.

Tibialdyschondroplasia (TD) is a disease characterized by the formation of an avascular, non-mineralized lesion along the mature face of the epiphyseal growth plate in rapidly growing chickens. In the normal growth plate, cells progress from a proliferative phase to hypertrophy where the tissue is vascularized and replaced by trabecular bone. In TD, cells apparently cease their development early in the transition to hypertrophy. These diseased cells are not removed by vascularization nor does mineralization occur. The resulting lesion increases in size as proliferative cells continue to divide in the absence of removal and replacement of cartilage by bone. This laboratory has previously reported that cells of the TD lesion have the morphological appearance of necrotic cells or in some cases apoptotic cells. In this study we examine in more detail the status of cells comprising the TD lesion using molecular techniques. Genomic DNA isolated from cells of severe TD lesions show the nucleosomal laddering indicative of apoptosis, while DNA isolated from proliferative and hypertrophic cells does not. This result was confirmed by the use of the Cell Death Detection ELISA which shows quantitatively that cells from severe TD lesions contain nearly twice as many nucleosomal fragments as cells from the hypertrophic zone while proliferative chondrocytes do not have significant fragmentation. In situ examination of the epiphyseal growth plate with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) clearly shows that the cells of the severe TD lesion are apoptotic. Cells from smaller lesions are stained to a lesser extent or not at all by TUNEL. We believe that the apoptosis seen in TD is a secondary effect of the disease and not its primary cause.

Animals↗

PubMed1996

Basic fibroblast growth factor: an autocrine growth factor for epiphyseal growth plate chondrocytes.

Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue.

Animals↗

PubMed1996

Immunolocalization of osteonectin in avian tibial dyschondroplastic cartilage.

Osteonectin is an acidic calcium-binding protein found in cartilage, bone matrix, vascular endothelium, and areas of tissue repair. Using immunocytochemistry, osteonectin has been localized in all zones of the normal avian epiphyseal growth plate with notably high amounts in the hypertrophic zone. In the proximal portion of this zone the staining was intracellular, while in the distal calcifying portion of the hypertrophic zone staining was both intracellular and extracellular. Osteonectin was also detected in the growth plate associated with lesions of chickens with tibial dyschondroplasia (TD). Intense intracellular staining was observed in hypertrophic chondrocytes proximal to the lesion; staining was markedly diminished in the TD lesion; extracellular matrix was devoid of staining. Staining intensity was high along the peripheral edges of the lesion that were undergoing vascularization and resorption. This was the only area in the dysplastic cartilage where staining was observed in the extracellular matrix as well as intracellularly. Similar patterns were viewed in all TD lesions examined, whether they were spontaneous or induced by dietary treatments or genetic selection.

Animals↗

PubMed1996

Immunolocalization of basic fibroblastic growth factor in avian tibial dyschondroplastic cartilage.

Recent research in our laboratory has demonstrated that basic fibroblast growth factor (bFGF) is a permissive mitogen for epiphyseal growth plate chondrocytes. Immunocytochemistry demonstrated the presence of bFGF in the proliferative and hypertrophic zones of normal epiphyseal growth plates of 4-wk-old broiler chickens. The purpose of this investigation was to extend this research to include examination of the status of bFGF in the cartilage lesion associated with tibial dyschondroplasia (TD). Immunocytochemistry revealed that the distribution of bFGF in the growth plate proximal to the TD lesion was similar to that observed with normal growth plate. However, the intensity of immunofluorescence was greatly diminished in the TD lesion. The number of chondrocytes staining positive for bFGF was also reduced. In the peripheral edges of the lesion where cartilage was being actively resorbed, the staining intensity was greatly increased when compared to the rest of the TD lesion. Similar patterns were observed in all TD tissues examined whether the lesions were spontaneous or induced by dietary treatments or genetic selection. It is hypothesized that the decrease in bFGF, a potent angiogenic factor, may be responsible for the poor vascularization of the TD lesion.

Animals↗

PubMed1995

Osteopontin gene expression in avian tibial dyschondroplasia.

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Animals↗