Fibulin-3 promotes muscle-invasive bladder cancer.

Urothelial carcinoma is the most common type of bladder cancer and can be categorized as either non-muscle-invasive (Ta-T1) or muscle-invasive (⩾T2). The majority of bladder cancers are non-muscle-invasive at presentation; however, the recurrence rate for these tumors is high and a subset can progress to T2. In this study, we aimed to identify genes differentially expressed between T1 vs T2 bladder cancer to identify key regulators of bladder cancer progression and/or invasion. We performed RNA-Seq on T1 and T2 bladder cancer tissues and used publicly available bladder cancer profiling studies to prioritize differentially expressed genes for validation and functional assessment. This integrative approach nominated an extracellular matrix glycoprotein, fibulin-3 (FBLN3, also known as EFEMP1), as being highly expressed in T2 vs T1 bladder cancer and aggressive vs indolent disease. We confirmed the overexpression of fibulin-3 in ⩾T2 vs non-muscle-invasive bladder cancer (NMIBC) by quantitative reverse transcriptase-PCR. Consistent with these findings, fibulin-3 expression level correlated with the invasive ability of several bladder cancer cell lines and modulation of fibulin-3 expression directly affected invasion. Fibulin-3 knockdown in bladder cancer cells decreased the incidence of MIBCs in a murine orthotopic bladder cancer model and decreased the expression of insulin-like growth factor-binding protein-5 (IGFBP5). Restoring IGFBP5 in these cells rescued their invasive and migratory potential. These results indicate that fibulin-3 serves as a pro-invasive factor in bladder cancer, which may be mediated through modulation of IGFBP5 expression. This also suggests fibulin-3 and IGFBP5 may have potential as biomarkers of aggressive bladder cancer or therapeutic targets.

The National Environmental Respiratory Center (NERC) experiment in multi-pollutant air quality health research: II. Comparison of responses to diesel and gasoline engine exhausts, hardwood smoke and simulated downwind coal emissions.

The NERC Program conducted identically designed exposure-response studies of the respiratory and cardiovascular responses of rodents exposed by inhalation for up to 6 months to diesel and gasoline exhausts (DE, GE), wood smoke (WS) and simulated downwind coal emissions (CE). Concentrations of the four combustion-derived mixtures ranged from near upper bound plausible to common occupational and environmental hotspot levels. An "exposure effect" statistic was created to compare the strengths of exposure-response relationships and adjustments were made to minimize false positives among the large number of comparisons. All four exposures caused statistically significant effects. No exposure caused overt illness, neutrophilic lung inflammation, increased circulating micronuclei or histopathology of major organs visible by light microscopy. DE and GE caused the greatest lung cytotoxicity. WS elicited the most responses in lung lavage fluid. All exposures reduced oxidant production by unstimulated alveolar macrophages, but only GE suppressed stimulated macrophages. Only DE retarded clearance of bacteria from the lung. DE before antigen challenge suppressed responses of allergic mice. CE tended to amplify allergic responses regardless of exposure order. GE and DE induced oxidant stress and pro-atherosclerotic responses in aorta; WS and CE had no such effects. No overall ranking of toxicity was plausible. The ranking of exposures by number of significant responses varied among the response models, with each of the four causing the most responses for at least one model. Each exposure could also be deemed most or least toxic depending on the exposure metric used for comparison. The database is available for additional analyses.

Truncation of the beta-catenin binding domain of E-cadherin precedes epithelial apoptosis during prostate and mammary involution.

A potential target of hormone action during prostate and mammary involution is the intercellular junction of adjacent secretory epithelium. This is supported by the long-standing observation that one of the first visible stages of prostate and mammary involution is the disruption of interepithelial adhesion prior to the onset of apoptosis. In a previous study addressing this aspect of involution, we acquired compelling evidence indicating that the disruption of E-cadherin-dependent adhesion initiates apoptotic programs during prostate and mammary involution. In cultured prostate and mammary epithelial cells, inhibition of E-cadherin-dependent aggregation resulted in cell death following apoptotic stimuli. Loss of cell-cell adhesion in the nonaggregated population appeared to result from the rapid truncation within the cytosolic domain of the mature, 120-kDa species of E-cadherin (E-cad(120)). Immunoprecipitations from cell culture and involuting mammary gland demonstrated that this truncation removed the beta-catenin binding domain from the cytoplasmic tail of E-cadherin, resulting in a non-beta-catenin binding, membrane-bound 97-kDa species (E-cad(97)) and a free cytoplasmic 35-kDa form (E-cad(35)) that is bound to beta-catenin. Examination of E-cadherin expression and cellular distribution during prostate and mammary involution revealed a dramatic reduction in junctional membrane staining that correlated with a similar reduction in E-cad(120) and accumulation of E-cad(97) and E-cad(35). The observation that E-cadherin was truncated during involution suggested that hormone depletion activated the same apoptotic pathway in vivo as observed in vitro. Based on these findings, we hypothesize that truncation of E-cadherin results in the loss of beta-catenin binding and cellular dissociation that may signal epithelial apoptosis during prostate and mammary involution. Thus, E-cadherin may be central to homeostatic regulation in these tissues by coordinating adhesion-dependent survival and dissociation-induced apoptosis.

Signaling network of paclitaxel-induced apoptosis in the LNCaP prostate cancer cell line.

OBJECTIVES: To attempt to identify the relationship of the key regulator molecules in paclitaxel-induced apoptosis using two metastatic cell lines: the human prostate carcinoma LNCaP line and the cervical carcinoma HeLa cell line. METHODS: Both LNCaP and HeLa cells were continuously exposed to clinically achievable concentrations of paclitaxel and observed for activation of programmed cell death as measured by cytotoxic dose-response curves, poly(adenosine diphosphate-ribose) polymerase cleavage, bcl-2 phosphorylation, and the activation of caspase-7 (interleukin-1 beta converting enzyme (ICE)-LAP3). RESULTS: Initially, we asked whether paclitaxel-induced bcl-2 phosphorylation is triggered by the spindle assembly checkpoint via an active cdc2 kinase-dependent pathway and whether phosphorylation of endogenous bcl-2 is the signal that activates cell death machinery. Paclitaxel-induced G2/M cell cycle arrest correlated with cdc2 kinase activity and bcl-2 phosphorylation. Olomoucin, a specific inhibitor of cyclin-dependent kinases, inhibited bcl-2 phosphorylation. On the basis of these studies, we then investigated whether bcl-2 was phosphorylated in a cell cycle-dependent fashion. Analysis of synchronized HeLa cells demonstrated that endogenous bcl-2 is phosphorylated in a G2/M cell cycle-dependent manner without apoptosis. CONCLUSIONS: Our results indicate that the events associated with paclitaxel-induced cytotoxicity are connected to each other and represent the signaling network of paclitaxel-induced mitotic arrest and cell death. In addition, we confirmed that the death-decision of paclitaxel-induced apoptosis is not mediated by bcl-2 phosphorylation and believe that this decision may be mediated by the activated spindle assembly checkpoint.

E-cadherin mediates aggregation-dependent survival of prostate and mammary epithelial cells through the retinoblastoma cell cycle control pathway.

E-cadherin and the retinoblastoma tumor suppressor (Rb) are traditionally associated with diverse regulatory aspects of cell growth and differentiation. However, we have discovered new evidence, which suggests that these proteins are functionally linked in a physiologic pathway required for cell survival and programmed cell death. Pharmacological activation of protein kinase C (PKC) or inducible overexpression and activation of the alpha isozyme of PKC (PKCalpha) resulted in approximately 60% apoptosis of mammary and prostate epithelial cells. Interestingly, the surviving cells had undergone dramatic aggregation concurrent with increased E-cadherin expression. When aggregation was inhibited by the addition of an E-cadherin-blocking antibody, apoptosis increased synergistically. We hypothesized that survival of the aggregated population was associated with contact-inhibited growth and that apoptosis might result from aberrant growth regulatory signals in non-aggregated, cycling cells. This hypothesis was confirmed by experiments that demonstrated that E-cadherin-dependent aggregation resulted in Rb-mediated G1 arrest and survival. Immunoblot analysis and flow cytometry revealed that hypophosphorylated Rb was present in non-aggregated, S phase cultures concurrent with synergistic cell death. We have also determined that the loss of membrane E-cadherin and subsequent hypophosphorylation of Rb in luminal epithelial cells preceded apoptosis induced by castration. These findings provide compelling evidence that suggests that E-cadherin-mediated aggregation results in Rb activation and G1 arrest that is critical for survival of prostate and mammary epithelial cells. These data also indicate that Rb can initiate a fatal growth signal conflict in non-aggregated, cycling cells when the protein is hypophosphorylated as these epithelial cells enter S phase.

Food and nutrient intake of a national sample of 4-year-old children in 1950: comparison with the 1990s.

OBJECTIVE: To evaluate the food and nutrient intake of members of a birth cohort study when young children in 1950 and investigate differences from present-day children's diets. DESIGN: One-day recall diet records from the MRC National Survey of Health and Development (NSHD) (1946 Birth Cohort) at age 4 years were analysed for energy and selected nutrients and compared to the published results for 4-year-olds in the 1992/93 National Diet and Nutrition Survey (NDNS). SETTING: England, Scotland and Wales in 1950 and 1992/93. SUBJECTS: 4,599 children in 1950 and 493 children in 1992/93. RESULTS: Mean (SD) daily intakes in 1950 were energy 1,445 (343) kcal, or 6.1 (1.4) MJ, protein 46 (11)g, fat 64 (20)g, starch 117 (33)g, sugar 62 (24)g, unavailable carbohydrate 13 (4)g, calcium 736 (230) mg, iron 7.7 (2.1) mg, retinol 738 (1,273) microg, carotene 1,049 (1,130) microg and vitamin C 40 (26) mg. Compared to 1992/93, the 1950 diet contained substantially more bread and vegetables and less sugar and soft drinks, giving it a higher starch and fibre content and making it more in line with current recommendations on healthy eating. However, fat provided 40% of energy in 1950, compared to 35% in 1992/93. In 1950, red meat was an important source of iron, but by 1992 most iron came from fortified breakfast cereals. Vitamin C came mainly from vegetables in 1950, but from soft drinks in 1992. CONCLUSIONS: The relative austerity of post-war food supplies resulted in food and nutrient intakes in 1950 which in many respects may well have been beneficial to the health of young children, despite fat intake being higher than present-day recommendations.

Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation: a potential early step in drug resistance.

The chemotherapeutic agent paclitaxel disrupts microtubule dynamics causing mitotic arrest, which leads to cell death. However, in paclitaxel-resistant tumor cells, treatment with paclitaxel induces abnormal progression through prophase resulting in a multimininucleated phenotype. Multimininucleation and subsequent polyploidization have been correlated with paclitaxel resistance. Paclitaxel treatment of HeLa cells resulted in cell death via typical activation of the apoptotic machinery, whereas treatment of the relative paclitaxel-resistant prostate cancer cell line PC-3 induced an attenuated caspase activation and multimininucleation. The multimininucleated phenotype could be mimicked in HeLa cells treated with paclitaxel and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a peptide caspase inhibitor. Interestingly, we observed no discernible difference in the pattern of cdc-2 kinase activation or phosphorylation of bcl-2-like proteins in PC-3 and HeLa cells treated with paclitaxel, which demonstrated that these molecules could not be used as indicators for the degree of caspase activation. In this study, we establish a connection between relative paclitaxel resistance, caspase attenuation/inhibition, and the multimininucleated phenotype.

Zinc and phytate intake of rural Gambian infants: contributions from breastmilk and weaning foods.

Zinc and phytate intakes of 183 rural Gambian infants were obtained from weighed records of breastmilk and food intake and measured contents in foods. Total zinc intake of 2.7 mg/d in the first month of age declined to 1.5 mg/d at 3 months, then increased to 4.3 mg/d by 17 months. Breastmilk was an important source of zinc, but the predominant cereal and groundnut-based foods had high [phytate]/[Zn] molar ratios ranging from 13 to 28, indicating potential impaired zinc bioavailability. The [phytate]/[Zn] molar ratio for the diet as a whole was low in early infancy, but increased to 13 in the second year. In contrast, this ratio was less than 6 for the diet of 48 Cambridge breastfed infants up to 18 months. A further disadvantage to the Gambian infants was indicated by their lower intake of protein of animal origin. However, calcium intake was estimated in both communities to be below the level which could give rise to zinc chelation in association with phytate. Compared to 'basal' and 'normative' requirements, total zinc intake of the Gambian infants showed the greatest shortfall between 3 and 12 months, making this the age band for maximum probable benefit from focused intervention programmes.

Retinoblastoma protein-dependent growth signal conflict and caspase activity are required for protein kinase C-signaled apoptosis of prostate epithelial cells.

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of 12-O-tetradecanoylphorbol-13-acetate in the prostate epithelial cell line, LNCaP, resulted in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, were resistant to apoptosis. LNCaP apoptosis was initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict was prevented by serum depletion, apoptosis was suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, afforded nearly total protection from protein kinase C-signaled apoptosis. This protection correlated with the total loss of caspase activity as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, we propose that protein kinase C regulates a novel cell death pathway that is initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells and that is executed by the caspase family of cysteine proteases.

Directional preferences in the rotational play behaviors of young children.

In a day-care setting, 33 girls and 34 boys, ranging in age from 36 to 73 months, responded to 30 tasks to obtain conventional measures of laterality and to determine their directional preferences on behaviors requiring whole body movement in a circle (circling) or pivoting about the body's vertical axis. Significant counterclockwise preferences were found for both kinds of turning behaviors, and there were no gender differences in these preferences. Increasing age was weakly associated with stronger right-handedness and counterclockwise preference on the circling tasks. These results are consistent with those of other studies and suggest that humans exhibit a small left-turning population preference in rotational movements.

Cell anchorage regulates apoptosis through the retinoblastoma tumor suppressor/E2F pathway.

Epithelial cells are dependent upon adhesion to extracellular matrix for survival. We show that loss of beta1 integrin receptor contact with extracellular matrix signals the inhibition of G1 cyclin-dependent kinase activity. This loss of cyclin-dependent kinase activity leads to accumulation of the hypophosphorylated (active) form of the retinoblastoma tumor suppressor protein (Rb). We present evidence that in epithelial cells deprived of matrix contact, the growth suppression signal elicited by hypophosphorylated Rb opposes stimulatory signals from serum growth factors, leading to a cell cycle conflict that triggers apoptosis. This apoptotic pathway is modulated by Bcl-2 through a novel mechanism that regulates Rb phosphorylation. We present evidence that the Rb-dependent apoptotic pathway functions in vivo in the apoptosis of the prostate glandular epithelium following castration.

The importance of the continued use of fluoride in the reduction of dental caries in children.

Fluoride continues to be a safe and effective public health measure. In spite of a recent decline in caries reduction in the US due to the large number of supplemental fluorides available, water fluoridation remains a cost-effective method to prevent decay in children. Also, a review of the literature shows a lack of ill effects associated with fluoride use in children. Most recently, the United States Public Health Service continues to support fluoridation in terms of policy and research. In 1990, the US toxicology Program concluded there were no detrimental effects including cancer risk from fluoride use.

The calcium and phosphorus intakes of rural Gambian women during pregnancy and lactation.

The Ca and P intakes of 148 pregnant and lactating women in a rural village in The Gambia, West Africa, have been estimated by direct weighing of food on a total of 4188 d. The Ca and P contents of local foods were determined by analysis of raw ingredients, snack foods and prepared dishes. Information about the contribution of mineral-rich seasonings was obtained. Efforts were made to discover unusual sources of Ca that might not be perceived as food by subject or observer. The main contributors to daily Ca intake were shown to be leaves, fish, cereals, groundnuts and local salt. Cow's milk accounted for only 5% of Ca intake. Unusual sources of Ca were discovered, namely baobab (Adansonia digitata) fruit and selected earths, but these were consumed infrequently and their contributions to Ca intakes were small. Cereals and groundnuts were the main sources of P. Ca and P intakes (mg/d) were shown to average 404 (SD 110) and 887 (SD 219) respectively. Seasonal changes in the availability of leaves, cereals and groundnuts resulted in variations in Ca and P intakes. The rainy season was associated with increased Ca intakes (by 16%) but decreased P consumption (by 15%). No difference was observed in Ca intake between pregnant and lactating women but P intake in lactation was 11% higher than that in pregnancy during the post-harvest season. The implications of these low Ca intakes require investigation.

Bacterial expression, purification, and partial characterization of amino acids 94-155 of human tissue factor pathway inhibitor (TFPI) as an inhibitor of blood coagulation factor Xa.

Tissue factor pathway inhibitor (TFPI) is a plasma-derived protein which inhibits two of the active serine proteases present during normal blood coagulation. Inhibition of both of these proteases, factors VIIa and Xa, is thought to require a factor Xa-TFPI complex. To begin to investigate the interactions between factor Xa and TFPI, amino acids 94-155, which encode for the second Kunitz domain (K2) of TFPI, were expressed, purified, and partially characterized. Expression of the recombinant peptide was accomplished using an E. coli expression system which produced the peptide at an expression level of approximately 2-5% of total cell protein. The peptide was localized to disulfide-linked refractile bodies which were solubilized by reduction in the presence of denaturant and the soluble protein refolded. Oxidized K2 was purified from the refold mixture using a two-step procedure employing gel filtration chromatography and reverse-phase HPLC. The unprocessed form of the recombinant peptide, Met-Ala-K2 (rMA-K2), was characterized. This peptide was purified to apparent homogeneity as determined by SDS-PAGE, quantitative amino acid, Edman degradation, and electrospray mass spectrometry analyses (> 95% pure). The product bound to factor Xa covalently coupled to a solid support in the presence of 2M sodium chloride demonstrating its affinity for this enzyme. Preincubation of rMA-K2 peptide with factor Xa neutralized, with 1.1:1 stoichiometry, the ability of factor Xa to hydrolyze a small chromogenic substrate. Additionally, rMA-K2 prolonged the time to clot formation in a plasma-based assay dependent on factor Xa concentration. Finally, this peptide mildly prolonged the prothrombin and modified prothrombin times of normal pooled plasma. Taken together this data demonstrates that this region of TFPI inhibits factor Xa activity and allows for further characterization of this enzyme-inhibitor complex.

Comparison of recombinant tissue factor pathway inhibitors expressed in human SK hepatoma, mouse C127, baby hamster kidney, and Chinese hamster ovary cells.

Recombinant tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK hepatoma and C127 rTFPIs (M(r) approximately 38,000) appear larger than those of BHK and CHO rTFPIs (M(r) approximately 35,000). All these proteins inhibit factor Xa and appear to bind factor Xa with 1:1 stoichiometry. The ability of these proteins to inhibit tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific antisera against the amino- and carboxy-termini of TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the protein. Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of TFPI occurs to different extent when TFPI is expressed in different cells and that the carboxy terminal region of the TFPI molecule is important for the inhibition of tissue factor-induced coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor.

BACKGROUND: This study was designed to determine whether arterial reocclusion after thrombolysis can be prevented by lipoprotein-associated coagulation inhibitor (LACI), a physiological inhibitor of tissue factor-induced coagulation mediated by the extrinsic pathway. METHODS AND RESULTS: Thrombosis was induced in femoral arteries of anesthetized dogs with the use of anodal current to elicit extensive vascular injury and formation of platelet-rich thrombi in one artery and with thrombogenic copper wire to elicit fibrin-rich thrombi without appreciable vascular injury in the contralateral artery. Recanalization of both vessels was induced with t-PA (1.7 mg/kg i.v. over 1 hour) and verified with Doppler flow probes. Reocclusion occurred within 2 hours in seven of seven arteries with electrical injury-induced thrombosis and in four of seven arteries with copper wire-induced thrombosis in the absence of LACI. In dogs given infusions of recombinant DNA-produced LACI (225 micrograms/kg over 15 minutes, followed by 4 micrograms/kg/min i.v.) after completion of the infusion of t-PA, no reocclusion occurred during the 2-hour interval of observation in any of the five arteries subjected to electrical injury (p less than 0.001), and cyclic partial occlusions were nearly abolished (0.4 +/- 0.4/hr in LACI-treated dogs compared with 13.7 +/- 5.5/hr in saline-treated dogs, p less than 0.0001). In contrast, reocclusion occurred in two of five arteries with indwelling copper wires, and cyclic partial occlusions were unaffected despite LACI. LACI prolonged the partial thromboplastin time modestly (1.7 +/- 0.2 x baseline) but did not affect platelet counts or aggregation assessed ex vivo. CONCLUSIONS: Inhibition of the extrinsic pathway of coagulation with LACI prevents thrombotic arterial reocclusion after thrombolysis in vessels subjected to extensive vascular injury. Our results demonstrate that activation of the extrinsic pathway plays a critical role in thrombotic reocclusion and that LACI provides a highly targeted approach to facilitate sustained recanalization without directly inhibiting platelets.

Recombinant lipoprotein-associated coagulation inhibitor inhibits tissue thromboplastin-induced intravascular coagulation in the rabbit.

Lipoprotein-associated coagulation inhibitor produces feed-back inhibition of tissue factor (tissue thromboplastin)-induced coagulation in the presence of factor Xa Recombinant lipoprotein-associated coagulation inhibitor (rLACI) was tested for its ability to modify thromboplastin-induced intravascular coagulation in a rabbit model that allows monitoring of iodine-125 fibrin accumulation/disappearance in the lung and sampling of blood for the measurement of coagulation parameters. Infusion of thromboplastin into the rabbit caused a rapid increase of radioactivity over the lungs, possibly due to the accumulation of 125I fibrin in the lungs, followed by a rapid decline of radioactivity, suggestive of removal of fibrin from the lungs. Thromboplastin also caused a rapid decrease of systemic fibrinogen that was accompanied by a lengthening of the activated partial thromboplastin time and prothrombin time. The effect of coinfusion of rLACI with thromboplastin or bolus injection of rLACI before thromboplastin infusion was studied. At a high dose of rLACI (800 micrograms/kg body weight), the thromboplastin-induced radioactivity increase in the lungs and the systemic fibrinogen decrease were completely suppressed. The activated partial thromboplastin time and prothrombin time of the plasma samples lengthened, possibly due to the presence of thromboplastin in circulation. The thromboplastin-induced radioactivity increase over the lungs was not completely suppressed by lower doses of rLACI (135 to 270 micrograms/kg body weight), but these doses of rLACI prevented systemic fibrinogen decrease. At a bolus dose of 23 micrograms/kg body weight, rLACI provided 50% protection of the fibrinogen consumption (fibrinogen decreased to 82% compared with 65% in rabbits treated with thromboplastin alone). These results show that rLACI is effective in the inhibition of thromboplastin-induced coagulation in vivo.

Immunoaffinity purification and characterization of lipoprotein-associated coagulation inhibitors from Hep G2 hepatoma, Chang liver, and SK hepatoma cells. A comparative study.

A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits. The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media. By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa. In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI. In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI. The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography. An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media. Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region. In addition, an Mr approximately 25,000 form of LACI was also present. The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay. To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation. LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution. These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma. Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent. The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs.

The use of heart rate monitoring in the estimation of energy expenditure: a validation study using indirect whole-body calorimetry.

1. A modified heart rate (HR) method for predicting total energy expenditure (TEE) was cross-validated against whole-body calorimetry (CAL). Minute-by-minute HR was converted to energy expenditure (EE) using individual calibration curves when HR exceeded a pre-determined 'FLEX' value designed to discriminate periods of activity. ('FLEX' HR was defined as the mean of the highest HR during rest and the lowest HR during the lightest imposed exercise.) Sedentary EE (below FLEX) was calculated as the mean EE during lying down, sitting and standing at rest. Sleeping EE was calculated as basal metabolic rate (BMR) predicted from standard equations. 2. Calibration curves of oxygen consumption v. HR for different postures at rest and during exercise were obtained for twenty healthy subjects (eleven male, nine female); mean r 0.941 (SD 0.04). The mean FLEX HR for men and women were 86 (SD 10) and 96 (SD 6) beats/min respectively. 3. Simultaneous measurements of HR and EE were made during 21 h continuous CAL, which included 4 x 30 min imposed exercise (cycling, rowing, stepping, jogging). HR exceeded FLEX for a mean of 98 (SD 41) min. Mean TEE by CAL (TEE.CAL) was 8063 (SD 1445) kJ. 4. The HR method yielded a mean non-significant underestimate in TEE (TEE.HR) of 1.2 (SD 6.2)% (range -11.4 to +10.6%). Regression of TEE.HR (Y) v. TEE.CAL (X) yielded Y = 0.868 X + 927 kJ, r 0.943, SE of the estimate 458 kJ, n 20. 5. The satisfactory predictive power and low cost of the method makes it suitable for many field and epidemiological applications.