Fluoroacetate-metabolizing pseudomonad isolated from Dichapetalum cymosum

A pseudomonad was isolated from the fluoroacetate-producing plant Dichapetalum cymosum (Hook) Engl. and identified as Pseudomonas cepacia. We established that this isolate was capable of growing in fluoroacetate-enriched solutions without any reduction in growth rate. Our isolate of P. cepacia was capable of defluorinating 2.69 mg of fluoroacetate per 10(9) cells per h. Fluoroacetate was degraded to CO2 at a rate of 23.53 ng/10(9) cells per h.     

Inoculum size as a factor limiting success of inoculation for biodegradation

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Inoculum size as a factor limiting success of inoculation for biodegradation.

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms.

Pseudomonas cepacia G4 degrades trichloroethylene (TCE) via a degradation pathway for aromatic compounds which is induced by substrates such as phenol and tryptophan. P. cepacia G4 5223 PR1 (PR1) is a Tn5 insertion mutant which constitutively expresses the toluene ortho-monooxygenase responsible for TCE degradation. In groundwater microcosms, phenol-induced strain G4 and noninduced strain PR1 degraded TCE (20 and 50 microM) to nondetectable levels (< 0.1 microM) within 24 h at densities of 10(8) cells per ml; at lower densities, degradation of TCE was not observed after 48 h. In aquifer sediment microcosms, TCE was reduced from 60 to < 0.1 microM within 24 h at 5 x 10(8) PR1 organisms per g (wet weight) of sediment and from 60 to 26 microM over a period of 10 weeks at 5 x 10(7) PR1 organisms per g. Viable G4 and PR1 cells decreased from approximately 10(7) to 10(4) per g over the 10-week period.     

Interaction of cblA/adhesin-positive Burkholderia cepacia with squamous epithelium

A highly transmissible strain of Burkholderia cepacia from genomovar III carries the cable pilin gene, expresses the 22 kDa adhesin (cblA +ve/Adh +ve), binds to cytokeratin 13 (CK13) and is invasive. CK13 is expressed abundantly in the airway epithelia of cystic fibrosis (CF) patients. We have now investigated whether binding of cblA +ve/Adh +ve B. cepacia to CK13 potentiates bacterial invasion and epithelial damage using bronchial epithelial cell cultures differentiated into either squamous (CK13-enriched) or mucociliary (CK13-deficient) epithelia. Three different B. cepacia isolates (cblA +ve/Adh +ve, cblA +ve/Adh -ve and cblA -ve/Adh -ve) showed minimal binding to mucociliary cultures, and did not invade or cause cell damage. In contrast, the cblA +ve/Adh +ve isolate, but not others, bound to CK13-expressing cells in squamous cultures, caused cytotoxicity and stimulated IL-8 release within 2 h. By 24 h, this isolate invaded and migrated across the squamous culture, causing moderate to severe epithelial damage. A specific antiadhesin antibody, which blocked the initial binding of the cblA +ve/Adh +ve isolate to CK13, significantly inhibited all the pathologic effects. Transmission electron microscopy of squamous cultures incubated with the cblA +ve/Adh +ve isolate, revealed bacteria on the surface surrounded by filopodia by 2 h, and within the cells in membrane-bound vesicles by 24 h. Bacteria were also observed free in the cytoplasm, surrounded by intermediate filaments containing CK13. These findings suggest that binding of B. cepacia to CK13 is an important initial event and that it promotes bacterial invasion and epithelial damage.     

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.     

Fourteen-year survival of Pseudomonas cepacia in a salts solution preserved with benzalkonium chloride.

A strain of Pseudomonas cepacia that survived for 14 years (1963 to 1977) as a contaminant in an inorganic salt solution which contained commercial 0.05% benzalkonium chloride (CBC) as an antimicrobial preservative, was compared to a recent clinical isolate of P. cepacia. Ammonium acetate was present in the concentrated stock CBC solution, and served as a carbon and nitrogen source for growth when carried over into the salts solution with the CBC. The isolate's resistance to pure benzalkonium chloride was increased step-wise to a concentration of 16%. Plate counts showed 4 x 10(3) colony-forming units per ml in the salts solution. Comparison of growth rates, mouse virulence, antibiotics resistance spectra, and substrate requirements disclosed no differences between the contaminant and a recently isolated clinical strain of P. cepacia. The results indicate that it is critical that pharmaceutical solutions containing benzalkonium chloride as an antimicrobial preservative be formulated without extraneous carbon and nitrogen sources or be preserved with additional antimicrobial agents.     

Fourteen-year survival of Pseudomonas cepacia in a salts solution preserved with benzalkonium chloride.

A strain of Pseudomonas cepacia that survived for 14 years (1963 to 1977) as a contaminant in an inorganic salt solution which contained commercial 0.05% benzalkonium chloride (CBC) as an antimicrobial preservative, was compared to a recent clinical isolate of P. cepacia. Ammonium acetate was present in the concentrated stock CBC solution, and served as a carbon and nitrogen source for growth when carried over into the salts solution with the CBC. The isolate's resistance to pure benzalkonium chloride was increased step-wise to a concentration of 16%. Plate counts showed 4 x 10(3) colony-forming units per ml in the salts solution. Comparison of growth rates, mouse virulence, antibiotics resistance spectra, and substrate requirements disclosed no differences between the contaminant and a recently isolated clinical strain of P. cepacia. The results indicate that it is critical that pharmaceutical solutions containing benzalkonium chloride as an antimicrobial preservative be formulated without extraneous carbon and nitrogen sources or be preserved with additional antimicrobial agents.     

Activity and stability of a recombinant plasmid-borne TCE degradative pathway in suspended cultures

The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min. mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h.     

Resistance plasmids in Pseudomonas cepacia 4G9.

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

Quorum-Sensing Signals and Quorum-Sensing Genes in Burkholderia vietnamiensis

Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many Proteobacteria including a clinical isolate of Burkholderia cepacia. The B. cepacia isolate produces low levels of octanoyl-HSL. We have examined an environmental isolate of Burkholderia vietnamiensis. This isolate produced several acyl-HSLs. The most abundant species was decanoyl-HSL. Decanoyl-HSL in B. vietnamiensis cultures reached concentrations in excess of 20 microM. We isolated a B. vietnamiensis DNA fragment containing a gene for the synthesis of decanoyl-HSL (bviI) and an open reading frame that codes for a putative signal receptor (bviR). A B. vietnamiensis bviI mutant did not produce detectable levels of decanoyl-HSL.     

Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice

Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, approximately threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, metacyclic forms of isolate 573, like the CL isolate, produced high parasitemia (>10(6) parasites per ml at the peak), killing approximately 40% of animals, whereas infection with isolate 587 resulted in low parasitemia (<10(5) parasites per ml), with zero mortality. On the fourth day post-inoculation, tissue sections of the mouse stomach stained with hematoxylin and eosin showed a four to sixfold higher number of epithelial cells infected with isolate 573 or CL than with isolate 587. The rate of intracellular parasite development was similar in all isolates. Mimicking in vivo infection, parasites were treated with pepsin at acidic pH and then assayed for their ability to enter HeLa cells or explanted gastric epithelial cells. Pepsin extensively digested gp90 from isolate 573 and significantly increased invasion of both cells, but had minor effect on gp90 or infectivity of isolates 587 and CL. The profile of g82 digestion was similar in isolates 573 and 587, with partial degradation to a approximately 70 kDa fragment, which preserved the target cell binding domain as well as the region involved in gastric mucin adhesion. Gp82 from CL isolate was resistant to pepsin. Assays with parasites recovered from the mouse stomach 2 h after oral infection showed an extensive digestion of gp90 and increased infectivity of isolate 573, but not of isolate 587 or CL. Our data indicate that T. cruzi infection in vitro does not always correlate with in vivo infection because host factors may act on parasites, modulating their infectivity, as is the case of pepsin digestion of isolate 573 gp90.     

Incidence of the cblA major subunit pilin gene amongst Burkholderia species

PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.     

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.     

A field investigation of intra-cervical insemination with reduced sperm numbers in gilts

A novel insemination catheter with a smaller polyurethane tip for deeper insertion into the cervix of gilts was compared with the conventional catheter. The novel catheter could be inserted 31.4 mm deeper than the conventional catheter into the gilt cervix, but the difference diminished with parity until the sixth parity when there was no difference in penetration depth between the catheters. In Experiment 1, cyclic gilts were inseminated upon display of oestrus (back pressure test) in the presence of a boar (0 h) and 24 h later. The control group (n = 300) were inseminated with 2 x 10(9) total spermatozoa and the treatment group (n = 300) with 1 x 10(9) total spermatozoa per inseminate, in both cases utilising the novel insemination catheter. No significant differences were observed for farrowing rate and litter size, the values of which were those expected for natural mating. In Experiment 2, 66 cyclic gilts were subjected to the same heat detection and service regime as for Experiment 1 but were served with <1 x 10(9) total sperm cells per inseminate using the new device. Conception rates and embryo counts were recorded. Conception rate declined with <500 x 10(6) spermatozoa, and number of embryos (a reflection of potential litter size) was significantly reduced. Use of the new catheter for gilts with 1 x 10(9) total sperm cells per inseminate will achieve commercially acceptable fertility and fecundity levels, and offer substantial commercial benefits with more rapid genetic gains.     

Uptake of free hemoglobin by rat liver parenchymal cells

Parenchymal cells isolated from rat liver are capable of taking up free hemoglobin. Uptake was saturable with a concentration for half-maximal velocity of 1.35 mg/ml (1.99 X 10(-5) M) hemoglobin. At a concentration of 0.088 mg/ml, the endocytic index for hemoglobin uptake was 4.5 microliters/h per mg of cell protein. This may be compared with the rate of fluid pinocytosis by these cells of 0.025 microliter/h per mg of cell protein (determined with yeast invertase as the marker). Free beta globin chains were also taken up with an endocytic index of 26.7 microliters/h per mg of cell protein at a beta chain concentration of 0.075 mg/ml. Hemoglobin inhibited the uptake of labeled beta globin. Hemoglobin-haptoglobin complex at a concentration of 0.12 mg/ml (as hemoglobin) was cleared at a rate of 0.89 microliter/h per mg cell protein and its uptake was also inhibited by free hemoglobin. We conclude that haptoglobin serves to conserve the iron of hemoglobin by preventing its renal clearance and not by promoting its hepatic uptake.