Sarcopenia in Men With Bone-Predominant Metastatic Castration-Resistant Prostate Cancer Undergoing Ra-223 Therapy.

INTRODUCTION: Osteosarcopenia is the progressive loss of musculoskeletal structure and functionality, contributing to disability and mortality. Despite complex interactions between bone and muscle, osteosarcopenia prevention and treatment in men with metastatic castration-resistant prostate cancer (mCRPC) focuses predominantly on bone health. It is unknown whether Radium-223 (Ra-223) therapy affects sarcopenia. METHODS: We identified 52 patients with mCRPC who had received Ra-223 and had a baseline plus ≥1 follow-up abdominopelvic CT scan. The total contour area (TCA) and averaged Hounsfield units (HU) of the left and right psoas muscles were obtained at the inferior L3 endplate, and the psoas muscle index (PMI) was calculated therefrom. Intrapatient musculoskeletal changes were analyzed across various time points. RESULTS: TCA and PMI gradually declined over the study period (P = .002, P = .003, respectively), but Ra-223 therapy did not accelerate sarcopenia, nor the decline of HU compared to the pre-Ra-223 period. The median overall survival of patients with baseline sarcopenia was numerically worse (14.93 vs. 23.23 months, HR 0.612, P = .198). CONCLUSIONS: Ra-223 does not accelerate sarcopenia. Thus, worsening muscle parameters in men with mCRPC undergoing Ra-223 therapy are attributable to other factors. Further research is needed to determine whether baseline sarcopenia predicts poor overall survival in such patients.

On the Breakage of High Aspect Ratio Crystals in Filter Beds under Continuous Percolation.

PURPOSE: This work details experimental observations on the effect of liquid flow percolating through packed beds of crystals to elucidate how the filtration pressure severely alters the size distribution and crystal shape. Pressure filtration is widely used in the pharmaceutical industry, and frequently results in undesired size distribution changes that hinder further processing. METHODS: The percolation methodology presented fixes fluid flow through a bed of crystals, resulting in a pressure over the bed. X-ray computed tomography (XCT) provided detailed observations of the bed structure. Detailed 2D particle size data was obtained using automated microscopy and was analysed using an in-house developed tool. RESULTS: Crystal breakage is observed when the applied pressure exceeds a critical pressure: 0.5-1 bar for ibuprofen, 1-2 bar for ß-L glutamic acid (LGA) and 2-2.5 bar for para amino benzoic acid (PABA). X-ray computed tomography showed significant changes in bed density under the applied pressure. Size analysis and microscope observations showed two modes of breakage: (i) snapping of long crystals and (ii) shattering of crystals. CONCLUSION: LGA and PABA have a similar breakage strength (50 MPa), ibuprofen is significantly weaker (9 MPa). Available breakage strength data may be correlated to the volumetric Gibbs free energy. Data from 12 and 35 mm bed diameters compares well to literature data in a 80 mm filter; the smaller, easy to operate percolation unit is a versatile tool to assess crystal breakage in filtration operations.

Role of variants rs5030717 and rs5030718 of TLR4 in the risk prediction of nephropathy, hypertension and dyslipidaemia in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus describes a metabolic disorder characterised by prolonged elevated blood glucose that brings a risk of developing microvascular and macrovascular disease. Several factors, such as dysregulation of the Toll-like receptor 4 (TLR-4), are reputed to contribute to the multiple pathophysiological disturbances responsible for impaired glucose homeostasis. We hypothesised that variants rs5030717 and rs5030718 of TLR4 are associated with diabetic nephropathy, hypertension and dyslipidaemia. MATERIAL & METHODS: We recruited 370 diabetics (122 with nephropathy, 119 with hypertension and 129 with dyslipidaemia) and 120 ethnicity matched healthy controls. TLR4 polymorphisms were evaluated using polymerase chain reaction followed by restriction fragment length polymorphism analysis. The genotyping data were compared between cases and controls using chi-square test and logistic regression analysis. RESULTS: Although there was no overall difference in the genotype frequencies of TLR4 rs5030717 in diabetes v controls, the genotype frequencies of diabetic dyslipidaemia cases compared with controls were different (p = 0.001). Overall, the rs5030718 GA and GG genotype frequencies in the entire diabetes cohort were different from those of the controls (p = 0.037), and the frequencies of diabetic nephropathy cases (p = 0.03) and diabetic dyslipidaemia cases were different (p = 0.001) compared with controls. There were no links with diabetic hypertension. CONCLUSION: TLR4 polymorphisms rs5030717 and rs5030718 may be useful in predicting those type 2 diabetics who are at risk of hypertension, nephropathy and/or dyslipidaemia.

Genetic polymorphisms in KCNJ11 (E23K, rs5219) and SDF-1ß (G801A, rs1801157) genes are associated with the risk of type 2 diabetes mellitus.

Background Type 2 diabetes mellitus (T2DM) is a global major health problem resulting from interaction of environmental and genetic factors, examples of the latter being KCNJ11 (coding for part of the ATP-sensitive potassium channel) and SDF-1ß (coding for chemokine CXCL12). Our case-control study was conducted to assess whether recessive, dominant or additive genotype model associations of KCNJ11 (E23K, rs5219) and SDF-1ß (G801A, rs1801157) were more strongly linked to type 2 diabetes. Subjects & Methods Genetic polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism. Alleles and genotype frequencies between 200 cases and 200 controls were determined and compared. Results The dominant (EE v EK + KK, p = 0.022) and additive (EK v EE + KK, p = 0.021) models, but not the recessive model (KK v EE + EK, p = 0.727) of KCNJ11 were linked to diabetes. Similarly, the dominant (GG v GA + AA, p < 0.001) and additive (AG v GG + AA, p=<0.001) models, but not the recessive model (AA v AG + GG, p = 0.430) of SDF-1ß were linked to diabetes. The A allele (p = 0.006) of SDF-1ß was protective against the risk of T2DM. Conclusion Both dominant and additive models in both KCNJ11 (E23K, rs5219) and SDF-1ß (G801A, rs1801157) genetic polymorphisms are significantly associated with type 2 diabetes.

Design and Synthesis of Curcumin-Like Diarylpentanoid Analogues as Potential Anticancer Agents.

BACKGROUND: Curcumin is a polyphenolic natural compound with multiple targets that used for the prophylaxis and treatment of some type of cancers like cervical and pancreatic cancers. Some recent patent for curcumin for cancer has also been reviewed. OBJECTIVE: In this study, ten new curcumin derivatives were designed and synthesized and their cytostatic activity evaluated against the Hela and Panc cell lines that some of them showed more activity than curcumin. METHOD: In the present study, a series of mono-carbonyl derivatives of curcumin were designed and prepared. The details of the synthesis and chemical characterization of the synthesized compounds are described. The cytostatic activities of the designed compounds are assessed in two different tumor cell lines using MTT test. RESULTS: In vitro screening for human cervix carcinoma cell lines (Hela) and pancreatic cell lines (Panc-1) at 24 and 48 hour showed that all the analogs possessed good activity against these tumor cell lines and compounds 5a, 5c and 6 with high potency can be used as a new lead compounds for the designing and finding new and potent cytostatic agents. Docking studies indicated that compound 5c readily binds the active site of human glyoxalase I protein via two strong hydrogen bonds engaging residues of Glu-99 and Lys-156. CONCLUSION: Our results are useful in guiding a design of optimized ligands with improved pharmacokinetic properties and increased of anti-cancer activity vs. the prototype curcumin compound.

Strengthening Biosecurity in Iraq: Development of a National Biorisk Management System.

Since 2004, the Republic of Iraq has undertaken a concerted effort to comply with all of its international obligations to prevent the proliferation and the use of chemical, biological, radiological, and nuclear (CBRN) weapons. A centerpiece of this effort is Iraq's development of a National Biorisk Management System. The Iraqi National Monitoring Authority (INMA), which is responsible for CBRN security and non-proliferation in Iraq, has played a key role in establishing this system. This article provides an overview of Iraq's international non-proliferation commitments, describes the legal and organizational steps it has taken to implement these commitments, and examines current initiatives to strengthen Iraq's biosecurity.

Prolylcarboxypeptidase independently activates plasma prekallikrein (fletcher factor).

Prolylcarboxypeptidase isoform 1 (PRCP1) is capable of regulating numerous autocrines and hormones, such as angiotensin II, angiotensin III, αMSH1-13, and DesArg(9) bradykinin. It does so by cleaving a C-terminal PRO-X bond. Recent work also indicates that the human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells through an uncharacterized mechanism. This study aims to identify PRCP1 binding interaction and cleavage site on PK. Recently, a cDNA encoding a novel splice variant of the human PRCP1 was identified. This isoform differed only in the N-terminal region of the deduced amino acid sequence. Using structural and functional studies, a combination of peptide mapping and site-directed mutagenesis approaches were employed to investigate the interaction of PRCP1 with PK. Three PRCP peptides, in decreasing order of potency, from 1) the N-terminus of the secreted protein, 2) spanning the opening of the active site pocket, and 3) in the dimerization region inhibit PRCP activation of PK on endothelial cells. Investigations also tested the hypothesis that PRCP cleavage site on PK is between its C-terminal Pro 637 (P(637)) and Ala 638 (A(638)). Recombinant forms of PK with C-terminal alanine mutagenesis or a stop codon is activated equally as wild type PK by PRCP. In conclusion, PRCP1 interacts with PK at multiple sites for PK activation. PRCP1 also enhances FXIIa activation of PK, suggesting that its activation site on PK is not identical to that of FXIIa.

Association of Glutathione-S-Transferase (GSTM1 and GSTT1) and FTO Gene Polymorphisms with Type 2 Diabetes Mellitus Cases in Northern India.

Type 2 diabetes mellitus (T2DM) is growing in an epidemic manner across the world and India has the world's largest number of diabetic subjects. The present study was carried out to investigate the association of glutathione-S-transferase (GSTM1, GSTT1) and fat mass and obesity associated (FTO) gene polymorphisms with T2DM patients and controls, and its role in increasing the susceptibility to T2DM. A total of 198 subjects (101 T2DM patients and 97 controls) participated in this study. GSTM1, GSTT1 and FTO gene polymorphisms in the patients and controls were evaluated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). We observed significant association of GSTM1 positive (p = 0.046) and GSTM1 null (p = 0.046) genotypes with T2DM, while no significant association was found with the FTO gene polymorphism in our study. It seems that the GSTM1 gene polymorphism can be a predictive marker for early identification of a population at risk of T2DM. The potential role of GST and FTO gene polymorphisms as a marker of susceptibility to T2DM needs further studies in a larger number of patients.

NMR-based metabonomics study of sub-acute hepatotoxicity induced by silica nanoparticles in rats after intranasal exposure.

Silica nanoparticles (SiO(2) NPs) are widely used commercially; however, their potential toxicity on human health has attracted particular attention. In the present study, the intranasal toxicological effect of 10nm and 80nm SiO(2) NPs (dosed at 150µg for 90 days) on rats was investigated using conventional approaches and metabonomics analysis of serum. Oxidative stress was measured by assessing Lipid peroxide (LPO) levels and enzymatic activities of Superoxide dismutase (SOD), Catalase (CAT), and Glutathione (GSH) levels in liver tissue homogenate. These biochemical observations were supplemented by histological examination of liver sections. SiO(2) NPs enhanced lipid peroxidation with concomitant reduction in SOD, CAT, and GSH content. In addition, SiO(2) NPs also produced alterations in hepatic histopathology. We also evaluated the effect of SiO(2) NPs on the activities of hepatic enzymes such as aminotransferases (ALT/AST) and alkaline phosphatase (ALP) which revealed significant increase in their activity when compared with control. Metabonomic profile of 90 days SiO(2) NPs treated rat sera exhibited significant increase in lactate, alanine, acetate, creatine and choline coupled with a considerable decrease in glucose level. These perturbations, on the whole, implicate impairment in tricarboxylic acid cycle and liver metabolism, which suggests that silica nanoparticles may have a potential to induce hepatotoxicity in rats.

Screening of C-kit gene Mutation in Acute Myeloid Leukaemia in Northern India.

BACKGROUND: Acute Myeloid Leukaemia (AML) is a cancer of blood-forming cells in bone marrow. C-kit gene is a Receptor Tyrosine Kinase class III (RTK) that is expressed by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. It is known that c-kit is a proto-oncogene and the activating c-kit mutations are likely to contribute in the development of leukaemia in humans. Exon 11 of c-Kit gene is the frequent site for mutations in different kinds of tumours. METHODS: In order to determine the frequency and prevalence of exon 11 mutations in 51 AML cases, we have done polymerase chain reaction-single-strand conformational polymorphism followed by direct DNA sequencing. RESULTS: The c-kit mutations in exon 11 were detected in 15.68% (8/51) in AML cases. We have detected totally ten missense mutations in eight AML cases those include Lys550Asn, Tyr568Ser, Ile571Leu, Tyr578Pro, Trp582Ser and Arg588Met and novel missense mutations at codons Ile563Lys and Val569Leu. Mutations at codons Ile571Leu and Trp582Ser was found in two independent cases. CONCLUSION: The presence of c-kit mutations in our study adds to investigative spectrum of AML cases. Since the c-kit mutations are seen in other malignancies, mutations in exon 11 of the c-kit gene might be involve in pathogenesis and represent useful predictive genetic marker in AML. Further studies in larger group of cases possibly will be required to determine the prognostic implications and to investigate how these mutations are co-related to the progression and pathogenesis of AML.

Protective potential of Bacopa monniera (Brahmi) extract on aluminum induced cerebellar toxicity and associated neuromuscular status in aged rats.

The present study attempts to assess the comparative effects of Bacopa monniera, (40 mg/kg body weight) and donepezil (2.5 mg/kg b. wt) on aluminum (100 mg / kg b. wt. of AlCl3) mediated oxidative damage in the cerebellum of aged rats (24 months) along with the associated dysfunctioning of neuromuscular coordination and motor activity. A significant decrease in the activities of antioxidant enzymes and increased total reacting oxygen species, lipid and protein peroxidation products observed in aluminum exposed rats. We observed that treatment with B. monniera extract restored the altered antioxidant enzyme activities more, when compared with donepezil. However, acetylcholinesterase showed similar effect both in donepezil and B. monniera treated groups. The content of aluminum was increased in all experimental groups, however, iron content was found increased in all groups except the B. monniera treated groups. Moreover, aluminum treated groups of rats exhibited significant changes in behavioral profiles but these changes were in both B. monniera and donepezil treated groups. The light microscopic and ultrastructural studies revealed damaged Purkinje's neurons and altered granular cell layer along with the increased accumulation of lipofuscin granules in aluminum treated animals. These changes were quite less pronounced in B. monniera group than that of donepezil and this may be due to the reduction of excess iron content by B. monniera. On the basis of our results it may be concluded that Al may be linked with cerebellar degeneration and neuromuscular disorders while Bacopa monniera extract helps in reversing these changes.

Identification of lipopolysaccharide binding site on high molecular weight kininogen.

Plasma kallikrein kinin system (KKS) activation along with its cellular receptors expression are increased after injury and in patients with septic shock, hypotensive bacteremia and rhesus monkey infected with Salmonella typhimurium. KKS signaling cascade is activated by activated factor XII (FXIIa, Hageman factor)- and prolylcarboxypeptidase (PRCP)-dependent pathways on endothelial cells. Among the many entities that comprise the KKS, high molecular weight kininogen (HK), a bradykinin precursor, is critical in the assembly and activation of this system. HK is primarily expressed in the liver and secreted into the bloodstream. The activation of the KKS influences the permeability of the endothelium by liberating bradykinin (BK) from HK. BK is a potent inflammatory peptide which stimulates constitutive bradykinin B2 and inducible B1 receptors to release nitric oxide and prostacyclin. Regardless of the triggers, PK can only be activated on HK bound to the artificial negatively charged or to cell membrane surfaces. Since LPS has a negatively charged moiety and the ability to induce inflammatory responses in human, we determined the interaction between LPS and HK. HKH19 (HK cell binding site) and heparin inhibited LPS binding to HK with IC(50)s of 15nM and 20 microg/ml, respectively. C1-inhibitor and N-acetylglucosamine glycan inhibited LPS binding to HK with IC(50)s of about 10 microg/ml and 10mM, respectively. This novel study underscores the implication of HK in infection. We propose that HKH19, heparin, and C1-inhibitor present therapeutic potential for the treatment of sepsis and hypotensive bacteremia.

Impact of operator on determining functional parameters of nuclear medicine procedures.

OBJECTIVE: The study was designed to assess the significance of the interoperator variability in the estimation of functional parameters for four nuclear medicine procedures. MATERIALS AND METHODS: Three nuclear medicine technologists with varying years of experience processed the following randomly selected 20 cases with diverse functions of each study type: renography, renal cortical scans, myocardial perfusion gated single-photon emission computed tomography (MP-GSPECT) and gated blood pool ventriculography (GBPV). The technologists used the same standard processing routines and were blinded to the results of each other. The means of the values and the means of differences calculated case by case were statistically analyzed by one-way ANOVA. The values were further analyzed using Pearson correlation. RESULTS: The range of the mean values and standard deviation of relative renal function obtained by the three technologists were 50.65 +/- 3.9 to 50.92 +/- 4.4% for renography, 51.43 +/- 8.4 to 51.55 +/- 8.8% for renal cortical scans, 57.40 +/- 14.3 to 58.30 +/- 14.9% for left ventricular ejection fraction from MP-GSPECT and 54.80 +/- 12.8 to 55.10 +/- 13.1% for GBPV. The difference was not statistically significant, p > 0.9. The values showed a high correlation of more than 0.95. Calculated case by case, the mean of differences +/- SD was found to range from 0.42 +/- 0.36% in renal cortical scans to 1.35 +/- 0.87% in MP-GSPECT with a maximum difference of 4.00%. The difference was not statistically significant, p > 0.19. CONCLUSION: The estimated functional parameters were reproducible and operator independent as long as the standard processing instructions were followed.

Overexpression of prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese hamster ovary cells.

Plasma prekallikrein (PK) complexes with its receptor, high-molecular-weight kininogen (HK), on human umbilical vein endothelial cells (HUVEC). When assembled on endothelial cells, PK is activated to plasma kallikrein independent of factor XIIa by the serine protease prolylcarboxypeptidase (PRCP, Km= 9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (J Biol Chem 277: 17962-17969, 2002) and produced as a recombinant protein (Blood 103: 4554-4561, 2004). To additionally confirm that human PRCP is a physiological PK activator, PRCP was overexpressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a cytomegalovirus promoter, and CHO recombinant PRCP was expressed as a fusion protein with COOH-terminal enhanced green fluorescence protein (EGFP). The presence of recombinant PRCP in transfected CHO cells was detected by real-time RT-PCR, immunoblot, and immunoprecipitation. PRCP mRNA and PK activation were two- to threefold higher in transfected than in control CHO cells. The increase in PRCP-induced PK activation in the transfected CHO cells paralleled the increase in PRCP antigen expression, as determined by anti-PRCP and anti-green fluorescence protein antibodies. PK activation of the transfected cells was blocked by small interfering RNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation (IC50= 0.01 and 7.0 mM, respectively). Localization of PRCP in intact cells observed via confocal microscopy and flow cytometry also confirmed overexpression of PRCP on the external membrane. These investigations independently confirm that PRCP is expressed on cell membranes and that PRCP expression increases PK activation.

Assembly of high molecular weight kininogen and activation of prekallikrein on cell matrix.

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

Factor XI assembly and activation on human umbilical vein endothelial cells in culture.

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and > or =7 microM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 microM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 microM, respectively. Alternatively, peptide T249-F260 of FXI's apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent Kd of 6.9 +/- 3.0 nM and Bmax of 13 +/- 2.6 x 10(6) sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XI-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.

Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells.

The cellular localization of human cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR), and gC1qR, high-molecular-weight kininogen (HK)-binding proteins on endothelial cells, was determined. CK1 was found on the external membrane of nonpermeabilized endothelial cells by immunoperoxidase staining, immunofluorescence, and transmission electron microscopy using immunogold. Human umbilical vein endothelial cells (HUVECs) had 7.2 +/- 0.2 x 10(4) specific CK1 membrane sites/cell by (125)I-F(ab')(2) anti-CK1 antibody binding. Flow cytometry studies confirmed the presence of CK1, uPAR, and gC1qR on HUVECs. On laser scanning confocal microscopy and transmission electron microscopy, CK1 and uPAR, but not gC1qR, colocalized on the cell surface of HUVECs. The HUVEC surface distribution of these proteins was distinctly different from that for von Willebrand factor. In competitive inhibition experiments, anti-CK1, anti-uPAR, or anti-gC1qR blocked both biotin-HK binding and prekallikrein (PK) activation on HUVECs with an inhibitory concentration of 50% (IC(50)) of 300 to 350 nM, 50 to 60 nM, or 35 to 100 nM, respectively. Also, antibodies to uPAR and gC1qR each inhibited 86% of kallikrein-mediated, 2-chain urokinase plasminogen activation, whereas antibodies to CK1 only inhibited 24% of plasminogen activation. On HUVECs, CK1 and uPAR, but not gC1qR, colocalized to be a multiprotein receptor complex for HK binding, PK activation, and 2-chain urokinase plasminogen activation.

Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation.

Prekallikrein (PK) activation on human umbilical endothelial cells (HUVEC) presumably leads to bradykinin liberation. On HUVEC, PK activation requires the presence of cell-bound high-molecular-weight kininogen (HK) and Zn(2+). We examined the Zn(2+) requirement for HK binding to and the consequences of PK activation on endothelial cells. Optimal HK binding (14 pmol/10(6) HUVEC) is seen with no added Zn(2+) in HEPES-Tyrode buffer containing gelatin versus 16--32 microM added Zn(2+) in the same buffer containing bovine serum albumin. The affinity and number of HK binding sites on HUVEC are a dissociation constant of 9.6 +/- 1.8 nM and a maximal binding of 1.08 +/- 0.26 x 10(7) sites/cell (means +/- SD). PK is activated to kallikrein by an antipain-sensitive mechanism in the presence of HK and Zn(2+) on HUVEC, human microvascular endothelial cells, umbilical artery smooth muscle cells, and bovine pulmonary artery endothelial cells. Simultaneous with kallikrein formation, bradykinin (5.0 or 10.3 pmol/10(6) HUVEC in the absence or presence of lisinopril, respectively) is liberated from cell-bound HK. Liberated bradykinin stimulates the endothelial cell bradykinin B2 receptor to form nitric oxide. Assembly and activation of PK on endothelial cells modulates their physiological activities.