Inhibition of osteoclast formation and function by bicarbonate: Role of soluble adenylyl cyclase

High inhibits and low stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM at pH 7.4 for 7–8 days and assayed for tartrate‐resistant acid phosphatase (TRAP) and vacuolar‐ATPase expression, and H⁺ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by in a dose‐dependent manner. V‐ATPase expression and H⁺ accumulation normalized to cell cross‐sectional area or protein were not significantly changed. The ‐induced inhibition of osteoclast growth and differentiation was blocked by either 2‐hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of inhibiting osteoclast via sAC was further supported by the fact that the dose‐response on osteoclasts is flat when cells were saturated with 8‐bromo‐cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1‐week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro‐computed tomography (µCT), was higher in 24 mM compared to 12 mM treated calvaria. This effect on BV/TV was blocked by 2‐hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by , by acting as a sensor. J. Cell. Physiol. 220: 332–340, 2009. © 2009 Wiley‐Liss, Inc.     

Adaptation of the rat cardiac proteome in response to intensity‐controlled endurance exercise

Endurance training improves cardiac function and protects against heart disease. The rodent intensity‐controlled running model replicates endurance exercise in humans and can be used to investigate molecular adaptations in the heart. Rats (n = 6, 280 ± 3 g) performed exercise tests to measure their peak oxygen uptake ( ) and training was prescribed at 70–75% for 30 min, 4 days/wk. Hearts were isolated 4 h after a final test and left ventricle proteomes compared to weight‐matched control animals (n = 6, 330 ± 2 g) using differential analysis of 2‐D gels. Proteins were identified by searching MS and MS/MS spectra against Swiss‐Prot using MASCOT (www.matrixscience.com). Average increased 23% (p = 0.008) over the 6‐week regimen and 23 gel spots differed (p<0.05) between exercised and control hearts. Expression of myofibrillar proteins (e.g. α‐myosin heavy chain and cardiac α‐actin) and proteins associated with fatty acid metabolism (e.g. heart fatty acid binding protein, acetyl coenzyme A dehydrogenase and mitochondrial thioesterase‐1) increased. In addition, this work discovered a novel increase in phosphorylation of heat shock protein 20 at serine 16. Previously this modification has been associated with improved cardiomyocyte contractility and protection against apoptosis.     

Component Analysis and Free Radical‐Scavenging Potential of Panax notoginseng and Carthamus tinctorius Extracts

Panax notoginseng and Carthamus tinctorius are known as traditional medicinal plants, and they also have edible values. To better understand their pharmacological mechanism, the present study assessed the in vitro antioxidant activities of extracts of P. notoginseng (EPN) and C. tinctorius (ECT). In addition, the main components of EPN and ECT were determined by HPLC. The results show that EPN mainly contained saponins, which were effective in scavenging ^.OH and O$\rm{{_{2}^{{^\cdot} -}}}$ , while showing a low activity in the DPPH^. assay. Flavonoids were the main components of ECT and were active in scavenging all three radicals in a dose‐dependent manner. In brief, the antioxidant properties of EPN and ECT are distinct and might be complementary, their combined use tending to be more effective in scavenging ^.OH (P<0.05 vs. EPN or ECT).     

Structure–activity relationship study of antioxidative peptides by QSAR modeling: the amino acid next to C‐terminus affects the activity

Screening, isolation and in vitro assays have been used for characterization of antioxidative peptides derived from food proteins, and incompatible deductions of structural characteristics derived from the isolated peptides have been brought forward. However, there is still little information concerning the structure‐activity relationship of antioxidative peptides. QSAR modeling was performed, respectively, on synthetic tripeptides and tetrapeptides related to LLPHH. According to cumulative squared multiple correlation coefficients (R²), cumulative cross‐validation coefficients (Q²) and relative standard deviation for calibration set (RSD_c), two credible models for tripeptide and tetrapeptide databases, respectively, have been built with partial least squares (PLS) regression (R² for models of tripeptide and tetrapeptide are 0.744 and 0.943, Q² are 0.631 and 0.414, and RSD_c are 0.323 and 0.111, respectively). Meanwhile, according to the cumulative multiple correlation coefficient for the predictive set ($R_{\rm {ext}}^{2}$ ) and the relative standard deviation for the predictive set (RSD_p), the predictive ability of the model for tripeptides also is excellent ($R_{\rm {ext}}^{2}$ and RSD_p are 0.719 and 0.450, respectively). Hydrogen bond property and hydrophilicity of the amino acid residue next to the C‐terminus, and the hydrophobicity as well as electronic propertyof the N‐terminus are more significant; meanwhile, the electronic property of the C‐terminus is beneficial for antioxidant activity. The structural characteristics we found are very useful in understanding and predicting the peptide structures responsible for activity and development of functional foods with peptides as active compounds, or antioxidative peptides as alternatives to other antioxidants. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Antimutagenic and Antioxidant Potentials of Teucrium ramosissimum Essential Oil

The mutagenic and antimutagenic effects of the essential oil extracted from the aerial parts of Teucrium ramosissimum were evaluated by the bacterial reverse mutation assay in Salmonella typhimurium TA98, TA100, and TA1535, with and without exogenous metabolic activation (S9 fraction). The T. ramosissimum essential oil showed no mutagenic effect. In contrast, our results established that it possessed antimutagenic effects against sodium azide (SA), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and 4‐nitro‐o‐phenylenediamine (NOPD). The antioxidant capacity of the tested essential oil was evaluated using enzymatic, i.e., the xanthine/xanthine oxidase (X/XOD) assay, and nonenzymatic systems, i.e., the nitro‐blue tetrazolium (NBT)/riboflavin and the DPPH assays. A moderate free radical‐scavenging activity was observed towards DPPH^. and O$\rm{{_{2}^{{^\cdot} -}}}$ . In contrast, T. ramosissimum essential oil showed no effect for all the tested concentrations in the X/XOD assay.     

Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.     

Antioxidant and Pro‐Oxidant Evaluation of a Potentilla alba L. Rhizome Extract

Using spectrophotometric methods, a H₂O‐soluble Potentilla alba L. rhizome extract was evaluated phytochemically, i.e., the total phenol, flavonoid, flavonol, flavanone, and proanthocyanidin contents were determined, and its antioxidant and pro‐oxidant properties, i.e., the Fe^III reductive and the Fe^II chelating properties, the 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH^.), N,N‐dimethyl‐p‐phenylenediamine (DMPD^.+), and superoxide anion radical (O$\rm{{_{2}^{{^\cdot} -}}}$ )‐scavenging activities, the capacity to inhibit hydroxyl radical (HO^.)‐mediated deoxy‐D ‐ribose and phospholipid degradation, and the interaction with the Cu‐catalyzed HO^.‐mediated DNA degradation, were determined. The extract was found to contain a range of phenolic compounds recognized to possess strong antioxidant‐like properties. Moreover, the extract demonstrated dose‐dependent activities in all the antioxidant assays with the exception of the DNA‐degradation assay, where the components within the extract interfered with the assay components at concentrations ≥1.00 mg/ml. Potentilla species are known for their curative properties, with aerial/subterranean parts being prescribed for numerous indications. The data presented here suggests, though does not conclude, that the rhizomes contain compounds possessing a range of antioxidant‐related properties, which may underpin the therapeutic, viz., anti‐inflammatory and adaptogenic effects, ascribed to species of this genus.     

Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is ${\hat {\mu }} = 0.42\pm 0.03\,{\rm h}^{- 1} $ even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of ${\hat {\mu }} = 0.62\pm 0.02\,{\rm h}^{- 1} $ . When in the same perfusion system cell‐free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to ${\hat {\mu }} = 0.47\pm 0.02\,{\rm h}^{- 1} $ . Likewise, when fresh medium is additionally supplied with 5 mM acetate, a growth rate of ${\hat {\mu }} = 0.51\pm 0.01\,{\rm h}^{- 1} $ is determined. These results prove that higher growth rates of C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely impaired by very low concentrations of byproducts such as acetate. Biotechnol. Bioeng. 2013; 110: 220–228. © 2012 Wiley Periodicals, Inc.     

The Unstirrable Water Layer Is Unstirrable because It Does Not Exist

A number of membrane‐permeation models require the incorporation of an unstirred or unstirrable water layer (UWL). An example occurs in PAMPA models when the effective permeation rate of lipophilic acids and bases, P_e, falls behind the expected permeation rate, P_m, at pH values providing a high concentration of unionized species in the donor phase. In such cases, the compound has an apparent pK_a of a weaker acid or base. The explanation is that an UWL adjacent to the membrane provides a rate‐limiting diffusion barrier for such compounds. The thickness of the UWL is correlated with the difference between the aqueous pK_a and the apparent pK_a (pK ). Here, we provide an explanation for the pK term that requires no UWL. It comes from the fact that, in the process of passing into a membrane, an ionizable compound undergoes a change in pK_a. At some point along its path into the membrane, the compound attains a maximum free energy, at which point it is as likely to continue into the membrane, as it is to return to the donor phase. This is the transition state for absorption. The pK is the pK_a of the compound at the transition state. This is a testable hypothesis (see text). The relevance of absorption to permeation depends on the rate‐limiting step of permeation.     

Garcimultiflorone G,a Novel Benzoylphloroglucinol Derivative from Garcinia multiflora with Inhibitory Activity on Neutrophil Pro‐Inflammatory Responses

A novel benzoylphloroglucinol derivative, garcimultiflorone G ( 1 ), was isolated from the fruits of Garcinia multiflora. The structure of 1 was determined through extensive 1D‐ and 2D‐NMR, and MS analyses. Garcimultiflorone G ( 1 ) showed inhibitory effects against superoxide anion (O$\rm{{_{2}^{{^\cdot} -}}}$ ) generation and elastase release by human neutrophils in response to formyl‐L ‐methionyl‐L ‐leucyl‐L ‐phenylalanine/cytochalasin B (fMLP/CB), with IC₅₀ values of 6.97±1.56 and 11.70±1.58 μM , respectively.     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

Ground‐ and excited‐state proton transfer and antioxidant activity of 3‐hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies

Excited‐state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3‐hydroxyflavone (3‐HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3‐HF against lipid peroxidation in this membrane system. When 3‐HF molecules are partitioned into EYPC liposomes, a weak long‐wavelength absorption band with λ ～410 nm appears in addition to the principal absorption at ～λ = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (λ～460 nm) of the ground‐state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (λ = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground‐state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3‐HF binding, suggesting that 3‐HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of calcium signaling pathways in human preadipocytes

Intracellular free Ca²⁺ (Ca) is an important regulator of many cellular activities; however, Ca²⁺ signaling is not well studied in human preadipocytes. The purpose of the present study was to characterize Ca²⁺ signal pathways using a confocal scanning technique and RT‐PCR. It was found that spontaneous Ca oscillations were observed in 12.1% preadipocytes, and number of cells with Ca²⁺ oscillations was increased to 47.9% by 1% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry mainly via stored‐operated Ca²⁺ (SOC) entry. They were suppressed by the SOC entry channel blocker La³⁺, the phospholipase C (PLC) inhibitor U73122, the inositol trisphosphate receptor (IP3R) blocker 2‐amino‐ethoxydiphenyl borate, or the sarcoplasmic/endoplasmic reticulum Ca²⁺ pump (SERCA) inhibitors thapsigargin and cyclopiazonic acid, but not by ryanodine. The IP3R activator thimerosal increased Ca oscillations. In addition, the plasma membrane Ca²⁺ pump (PMCA) inhibitor carboxyeosin and Na⁺–Ca²⁺ exchanger (NCX) inhibitor Ni²⁺ both suppressed Ca²⁺ oscillations. RT‐PCR revealed that the mRNAs for IP3R1‐3, SERCA1,2, NCX3 and PMCA1,3,4, Ca_V1.2, and TRPC1,4,6, STIM1 and Orai1 (for SOC entry channels) were significant in human preadipocytes. The present study demonstrates that multiple Ca²⁺ signal pathways are present in human preadipocytes, and provides a basis for investigating how Ca²⁺ signals regulate biological and physiological activities of human preadipocytes. J. Cell. Physiol. 220: 765–770, 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis,Structure, DNA‐Binding Properties,and Cytotoxicity of Ruthenium(II) Polypyridyl Complexes

Many ruthenium(II) complexes show high antitumor activities, and the in vitro antitumor activities are usually related to DNA binding. We designed and synthesized two Ru^II polypyridyl complexes, [Ru(dmp)₂(fpp)]²⁺ (dmp=2,9‐dimethyl‐1,10‐phenanthroline; fpp=2‐[3,4‐(difluoromethylenedioxy)phenyl]imidazo[4,5‐f] [1,10]phenanthroline and [Ru(phen)₂(fpp)]²⁺ (phen=1,10‐phenanthroline). The DNA‐binding properties of these complexes have been investigated by spectroscopic titration, DNA melting experiments, viscosity measurements, and photoactivated cleavage. The mechanism studies of photocleavage revealed that singlet oxygen (¹O₂) and superoxide anion radical (O$\rm{{_{2}^{{^\cdot} -}}}$ ) may play an important role in the photocleavage. The cytotoxicity of complexes 1 and 2 have been evaluated by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) method; complex 2 shows slightly higher anticancer potency than 1 does against all the cell lines screened.     

Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high‐affinity range of concentrations

The relative contribution of the high‐affinity K⁺ transporter AtHAK5 and the inward rectifier K⁺ channel AtAKT1 to K⁺ uptake in the high‐affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col‐0). The results obtained with wild‐type lines, with T‐DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the ‐sensitive and the Ba²⁺‐sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high‐affinity range of Rb⁺ concentrations. Using Rb⁺ as a K⁺ analogue, it was shown that AtHAK5 mediates absorption at lower Rb⁺ concentrations than AtAKT1 and depletes external Rb⁺ to values around 1 μM. Factors such as the presence of K⁺ or during plant growth determine the relative contribution of each system. The presence of in the growth solution inhibits the induction of AtHAK5 by K⁺ starvation. In K⁺‐starved plants grown without , both systems are operative, but when is present in the growth solution, AtAKT1 is probably the only system mediating Rb⁺ absorption, and the capacity of the roots to deplete Rb⁺ is reduced.     

The role of denitrification on arsenite oxidation and arsenic mobility in an anoxic sediment column model with activated alumina

Arsenite (As(III)) is the predominant arsenic (As) species in reducing environments. As(III) is less strongly adsorbed than As(V) at circumneutral pH conditions by common non‐iron metal oxides in sediments such as those of aluminum. Therefore, oxidation of As(III) to As(V) could contribute to an improved immobilization of As and thus help mitigate As contamination in groundwater. Microbial oxidation of As(III) is known to readily under aerobic conditions, however, the dissolved oxygen (O₂) concentration in groundwater may be limited due to the poor solubility of O₂ and its high chemical reactivity with reduced compounds. Nitrate (${\rm NO}_{3}^{{-} } $ ), can be considered as an alternative electron acceptor, which can support oxidation of As(III) to As(V) by denitrifying bacteria. In this study, two up‐flow sediment columns packed with activated alumina (AA) were utilized to demonstrate the role of denitrification on the oxidation of As(III) to As(V) and its contribution to improved As adsorption onto AA. One column was supplied with ${\rm NO}_{3}^{{-} } $ (C1) and its performance was compared with a control column lacking ${\rm NO}_{3}^{{-} } $ (C2). During most of the operation when the pH was in the circumneutral range (days 50–250), the release of arsenic was greater from C2 compared to C1. The effluent As concentrations started increasing on days 60 and 100 in C2 and C1, respectively. Complete breakthrough started on day 200 in C2; whereas in C1, complete breakthrough was never achieved. The effluent and solid phase As speciation was dominated by As(V) in C1, indicating the occurrence of As(III) oxidation due to ${\rm NO}_{3}^{{-} } $ ; whereas in C2, only As(III) was dominant. This study illustrates a bioremediation or natural attenuation process based on anoxic microbial ${\rm NO}_{3}^{{-} } $ ‐dependent oxidation of As(III) to more readily adsorbed As(V) as a means to enhance the immobilization of As on alumina oxide particles in subsurface environments. Biotechnol. Bioeng. 2010;107: 786–794. © 2010 Wiley Periodicals, Inc.     

Bayesian Bootstrap Clones for Censored Markov Chains

In this article, we consider r observations from a non‐homogeneous censored Markov chain, with transition probability matrix P. For the product estimator of P proposed by Aalen and Johansen (1978) and Phelan (1988), we investigate the behavior of Bayesian bootstrap clones to approximate the sampling distribution of , and then construct approximate confidence interval. It is shown that the approximation based on the random‐weighted distribution is first‐order consistent. The performance of the Bayesian bootstrap clones (BBC) is also discussed by small sample simulation. Finally, we illustrate the BBC procedure in the application to the WHO malaria survey data (cf. Singer and Cohen 1970).