Drag reduction by Acinetobacter calcoaceticus BD4

The encapsulated bacterium Acinetobacter calcoaceticus BD4 at a density of 3.6 X 10(9) cells per ml reduced the friction of turbulent water in a narrow pipe by 55%. This drag reduction was due to the tightly bound polysaccharide capsules (0.4 mg per ml) of culture. Capsule-deficient mutants of BD4 failed to reduce drag. The cell-bound polysaccharide demonstrated a threefold-higher drag-reducing activity than the polymer which was free in solution.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein.

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

灵芝液体发酵条件的研究

本文研究了某些因素对灵芝液体发酵的影响。4天菌龄的液体菌种发酵效果最好;300毫升三角瓶以装入60毫升培养液发酵较合适;蔗糖、花生饼粉分别为灵芝液体发酵的理想碳、氮源;液体发酵培养基最佳配方为:蔗糖4％、花生饼粉3％、硫酸铵0.15％、磷酸二氢钾0.15％,可得灵芝菌丝体百分干重为0.83,每100毫升发酵液得粗多糖0.3克。     

Drag-reducing polysaccharides from marine microalgae: species productivity and drag reduction effectiveness

We have conducted a study of the potential use of drag-reducing biopolymers produced by marine microalgae for engineering applications. Several marine microalgae species were tested for their production of drag-reducing polysaccharides in large custom-designed plate bioreactors. Promising species (such as Porphyridium cruentum, Rhodella maculata, Schizochlamydella capsulata and Chlorella stigmatophora) were cultured for periods of time ranging from a few weeks to over 6 months. The basic drag-reducing ability of the polysaccharides was established by comparing their drag reduction effectiveness at various concentrations in water. The algal polysaccharide mass productivity was also measured per unit area of bioreactor’s illuminated surface. Finally, an all-inclusive criterion, the volumetric production of drag-reducing water giving a set level of drag reduction was quantified, and led us to a ranking of the tested species in order of productivity relevant to implementation. Some aspects of polysaccharide production by aged cultures were investigated as well. We also quantified the drag-reducing effectiveness of intracellular polysaccharides, and visualized the presence of exopolymer particles in the medium.     

灵芝胞外多糖的发酵条件及其组份的初步研究

本文报道了液体培养灵芝Gl8801菌林产生胞外多糖的最佳发酵条件和胞外多搪的化学组成。适宜的液体培养基(g/L):饴糖40,花生饼粉30,KH_2PO_41.5,(NH_4)_2SO_41.5,MgSO_4·7H_2O_(0.75),CaCO_32,最适起始pH5.5。300ml三角瓶装培养基60ml,发酵温度28℃。摇瓶(165rpm)培养7～8天,发酵液纯多糖含量可达880mg/L。灵芝胞外多糖分为水溶性多糖和水不溶性多糖两种。水溶性多糖的单糖组份为半乳糖、葡萄糖、阿拉伯糖和木糖,所含单糖重量比为6:4:4:1。不溶性多糖为葡聚糖。灵芝胞外多糖的糖环构型为吡喃糖,末端糖基构型,半乳糖为α型,葡萄糖为β型。     

Methionine-hydroxy analogue was found to be significantly less bioavailable compared to dl-methionine for protein deposition in growing pigs

When methionine (Met) is limiting in swine diets, it is commonly supplemented by using anhydrous dl-methionine (DLM, 99% purity) or liquid dl-methionine-hydroxy analogue free acid (MHA-FA, 88% purity). The objective of this experiment was to test the null hypothesis that the bioavailability of DLM and MHA-FA were not different for growing pigs, using the indicator amino acid (AA) (phenylalanine, Phe) oxidation (IAAO) method in a slope-ratio assay. Six barrows (mean BW during study: 21.1 kg) received seven dietary treatments with all pigs receiving all diets in random order at an intake of 95 g/kg BW0.75. The basal diet (BD) contained analyzed content of 15.1% CP, 0.20% Met, 0.73% Phe and all other AA in excess of requirement. The BD was supplemented with three graded levels of DLM or MHA-FA on an equimolar basis. Dietary treatments only varied in Met content and included: (i) BD, (ii) BD + 0.034% DLM, (iii) BD + 0.054% DLM, (iv) BD + 0.086% DLM, (v) BD + 0.029% MHA-FA, (vi) BD + 0.078% MHA-FA and (vii) BD + 0.107% MHA-FA, as analyzed. Indicator AA oxidation was determined during 4 h studies, where pigs were fed half-hourly meals each equal to 1/32 of their daily feed allowance. Each meal was mixed with 258.7 kBq (s.e. 2.6) of l-[1-14C]Phe with a prime of 3.5 times the half-hourly dose added to the first meal. The slope of the decrease in IAAO calculated by linear regression analysis was greater (P = 0.012) for DLM supplementation (9.87 ± 1.450 per g, 1.488 ± 0.215% per mmol) than for MHA-FA (6.48 ± 0.89 per g, 1.107 ± 0.152% per mmol). The ratio of slopes indicated a bioavailability of MHA-FA on a product basis, relative to DLM, of 65.7%. Bioavailability on an equimolar Met basis, calculated from the ratio of the slopes was 74.4% for MHA-FA, relative to DLM. In conclusion, these results indicate that the metabolic bioavailability of MHA-FA for growing pigs is appreciably lower than that of DLM on both an equimolar and a product basis.     

Influence of flow on pulmonary vascular surface area inferred from blue dextran efflux data.

Blue dextran (BD), which binds to proteins on the pulmonary endothelial surface and to plasma albumin, was used in isolated perfused dog lung lobe experiments to address the question: do changes in perfusate flow rate cause changes in perfused vascular surface area? When BD was added to a protein-free perfusate under zone 3 conditions at a high flow rate (15.8 +/- 0.7 ml/s), it was adsorbed by the endothelial surface. Then by changing the perfusate entering the lobe to an albumin-containing perfusate, the BD was eluted from the perfused surface by competitive binding to the perfusate albumin. The amount of BD eluted was measured in three experiments. In experiment 1, elution of the BD by the perfusate albumin was initiated after a balloon had been inflated within the lobar arterial tree to occlude a portion of the lobar vascular bed containing BD. Then the balloon was deflated, permitting albumin perfusate to perfuse the previously occluded part of the lobe. In experiment 2, BD elution began at a flow rate of 3 +/- 0.1 ml/s under zone 3 conditions and continued after the high-flow zone 3 conditions were reestablished. In experiment 3, the BD elution began at a flow rate of 4.2 +/- 0.7 ml/s under zone 2 conditions and continued after the high-flow zone 3 conditions were reestablished. Balloon inflation reduced the amount of BD recovered by 43%, demonstrating that a decrease in perfused vascular surface area could decrease BD recovery.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,3-Butadiene and leukemia among synthetic rubber industry workers: exposure-response relationships

Previous research updated the mortality experience of North American synthetic rubber industry workers during the period 1944-1998, determined if leukemia and other cancers were associated with several employment factors and carried out Poisson regression analysis to examine exposure-response associations between estimated exposure to 1,3-butadiene (BD) or other chemicals and cancer. The present study used Cox regression procedures to examine further the exposure-response relationship between several unlagged and lagged, continuous, time-dependent BD exposure indices (BD parts per million (ppm)-years, the total number of exposures to BD concentrations >100 ppm ("peaks") and average intensity of BD) and leukemia, lymphoid neoplasms and myeloid neoplasms. All three BD exposure indices were associated positively with leukemia. Using continuous, untransformed BD ppm-years the regression coefficient (beta) from an analysis that controlled only for age was 2.9 x 10(-4) (p<0.01); the regression coefficient adjusted for all covariates (age, year of birth, race, plant, years since hire and dimethyldithiocarbamate) was similar in magnitude (beta=3.0 x 10(-4), p=0.04). Lagging exposure had minimal impact on the results for leukemia for any of the three BD exposure indices. In models that controlled only for age, lymphoid neoplasms were associated with BD ppm-years and myeloid neoplasms, with BD peaks, but neither trend was statistically significant after adjusting for multiple covariates. The present results support the presence of a causal relationship between high cumulative exposure and high intensity of exposure to BD and leukemia.     

Serum levels of TNF-alpha,sIL-2R,IL-6, and IL-8 are increased and associated with elevated lipid peroxidation in patients with Behçet's disease

AIM: Behçet''s disease (BD) is asystemic immunoinflammatory disorder and the aetiopathogenesis is to be specified. Cytokines play a role in immune response and in many inflammatory diseases. The aim of this case-control study is to investigate serum pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha, interleukin-1beta (IL-1beta), soluble IL-2 receptor (sIL-2R), IL-6, and chemokine IL-8 levels in patients with BD. We also determined the end product of lipid peroxidation (malondialdehyde (MDA)) in BD patients as an index for oxidative stress. METHODS: A total of 37 patients (19 men, 18 women) with BD (active, n = 17; inactive, n = 20) and 20 age-matched and sex-matched healthy control subjects (11 men, nine women) included in this cross-sectional, blinded study. Serum TNF-alpha, IL-1beta, sIL-2R, IL-6 and IL-8 levels were determined by a spectrophotometer technique using the immulite chemiluminescent immunometric assay. Lipid peroxidation was evaluated by Wasowicz et aL The levels of cytokines and lipid peroxidation in the active period were compared with the inactive period of the disease. Results are expressed as mean +/- standard error. RESULTS: IL-1beta levels were below the detection limits of the assay (< 5 pg/ml) in all samples. Mean levels of MDA (8.1+/-0.7 micromol/l), sIL-2R (800+/-38 U/ml), IL-6 (12.6+/-1.1 pg/ml), IL-8 (7.2+/-0.4 pg/ml), and TNF-alpha (7.9+/-0.5 pg/ml) in active BD patients were significantly higher than those in inactive patients (4.3+/-0.5 micromol/l, p < 0.01; 447+/-16 U/ml, p < 0.001; 8.3+/-0.6 pg/ml, p = 0.006; 5.3+/-0.1 pg/ml, p < 0.001; and 5.1 0.2 pg/ml, p < 0.001; respectively) or control subjects (2.1+/-0.2 micromol/l, p < 0.001; 446+/-20 U/ml, p < 0.001; 6.4+/-0.2 pg/ml, p < 0.001; 5.4+/-0.1 pg/ml, p < 0.001; and 4.7+/-0.1 pg/ml, p < 0.001, respectively). On the contrary, only the mean IL-6 level was significantly different between inactive BD and control subjects (p = 0.02). All acute phase reactants were significantly higher in active BD than in inactive period (for each, p < 0.01). Conclusions: High levels of sIL-2R, IL-6, IL-8 and TNF-alpha indicate the activation of immune system in BD. Serum sIL-2R, IL-6, IL-8 and TNF-alpha seem to be related to disease activity. Increased lipid peroxidation suggests oxidative stress in BD and therefore tissue damage in such patients. Amelioration of clinical manifestations would be envisaged by targeting these cytokines, chemokines and lipid peroxidation with pharmacological agents.     

Structures of Cinnzeylanine and Cinnzeylanol,Polyhydroxylated Pentacyclic Diterpenes from Cinnamonum zeylanicum Nees

A bacterium isolated as resistant to alkyldimethylbenzylammonium chloride (benzalkonium chloride, BC) and tentatively identified as Enterobacter cloacae, was induced by BC to produce acidic polysaccharide. The optimum concentration of BC for production of the polysaccharide was 0.1% and the polysaccharide produced amounted to 1.0-2.0 mg per ml of culture broth. The best carbon and nitrogen sources for the polysaccharide production were glycerol and polypeptone.

The acidic polysaccharide was consisted of fucose, galactose, glucose, glucuronic acid, pyruvate, and acetate, like colanic acid. The production of the acidic polysaccharide was not induced by the addition of trimethylbenzylammonium chloride and tetramethylammonium chloride, but it was induced by p-fluorophenylalanine, and the results are discussed.     

Structural studies of the capsular polysaccharide of Acinetobacter calcoaceticus BD4

Compositional analysis of the intact and carboxyl-reduced capsular polysaccharide of Acinetobacter calcoaceticus BD4 (PS-4) showed it to consist of L-rhamnose, D-glucose, D-glucuronic and D-mannose in molar ratios of 4:1:1:1. 13C-nuclear magnetic resonance spectroscopy, methylation analysis, oligosaccharide analysis and base-catalyzed beta-elimination were used to elucidate the primary structure. Oligosaccharides were obtained by enzymatic depolymerization with a specific bacteriophage-induced depolymerase and by partial acid hydrolysis. Form the results it is concluded that PS-4 consists of repeating units of the heptasaccharide (Formula: see text). The bacteriophage-induced depolymerase was found to be an endo-beta-D-glucosidase that hydrolyzed the bond beta-D-Glc-(1----3)-L-Rha to generate a heptasaccharide in 40% yield.     

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6 microg/ml were much lower than the cytotoxic concentration 50% (CC50) values (1000 microg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60-100 microg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160 kDa, contained a backbone of alpha-(1-->3)-linked D-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of beta-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of alpha-(1-->3)-linked D-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.     

Antiviral property and mechanisms of a sulphated polysaccharide from the brown alga Sargassum patens against Herpes simplex virus type 1

A sulphated polysaccharide (SP-2a) from the brown alga Sargassum patens (Kütz.) Agardh (Sargassaceae) was found to significantly inhibit the in vitro replication of both the acyclovir (ACV)-sensitive and -resistant strains of Herpes simplex virus type 1 (HSV-1), in dose-dependent manners, with 50% inhibitions occurring with 1.5–5.3 μg/ml of the polysaccharide. SP-2a exhibited extracellular virucidal activity only against the ACV-sensitive strains, but not the resistant strain, at the concentration of 100 μg/ml. The strongest antiviral activities against the different strains of HSV-1 were observed when this polysaccharide was present during and after adsorption of the virus to host cells. The inhibitory effect of SP-2a on virus adsorption occurred dose-dependently in all the HSV-1 strains tested, and the adsorption of the ACV-resistant DM2.1 strain was reduced by 81.9% (relative to control) with 4 μg/ml of the polysaccharide. This study clearly demonstrated that the antiviral mode of action of SP-2a is mediated mainly by inhibiting virus attachment to host cells, and this sulphated polysaccharide might have different modes of action against the ACV-sensitive and -resistant strains of HSV-1.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.     

Bubble rise velocities and drag coefficients in non-Newtonian polysaccharide solutions.

Microbially produced polysaccharides have properties which are extremely useful in different applications. Polysaccharide producing fermentations start with liquid broths having Newtonian rheology and end as highly viscous non-Newtonian solutions. Since aerobic microorganisms are used to produce these polysaccharides, it is of great importance to know the mass transfer rate of oxygen from a rising air bubble to the liquid phase, where the microorganisms need the oxygen to grow. One of the most important parameters determining the oxygen transfer rate is the terminal rise velocity of air bubble. The dynamics of the rise of air bubbles in the aqueous solutions of different, mostly microbially produced polysaccharides was studied in this work. Solutions with a wide variety of polysaccharide concentrations and rheological properties were studied. The bubble sizes varied between 0.01 mm3 and 10 cm3. The terminal rise velocities as a function of air bubble volume were studied for 21 different polysaccharide solutions with different rheological properties. It was found that the terminal velocities reached a plateau at higher bubble volumes, and the value of the plateau was nearly constant, between 23 and 27 cm/s, for all solutions studied. The data were analyzed to produce the functional relationship between the drag coefficient and Reynolds number (drag curves). It was found out that all the experimental data obtained from 21 polysaccharide solutions (431 experimental points), can be represented by a new single drag curve. At low values of Reynolds numbers, below 1.0, this curve could be described by the modofoed Hadamard-Rybczynski model, while at Re > 60 the drag coefficient was a constant, equal to 0.95. The latter finding is similar to that observed for bubble rise in Newtonian liquids which was explained on the basis of the "solid bubble" approach.     

Degradation of lignocellulose by extracellular enzymes produced by Thermomonospora fusca BD25

Degradation products from the addition of extracellular enzymes from Thermomonospora fusca BD25 to ball-milled wheat straw, oat spelt xylan and solubilised kraft pulps were characterised by HPLC and TLC. Overall, a high percentage hydrolysis of oat spelt xylan (28.9%) occurred after 26 h incubation. However, the rates of hydrolysis of ball-milled wheat straw and kraft pulp were approximately 4-6-fold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate was similar (22.2% and 25.9% respectively). Incubation of kraft pulp and ball-milled wheat straw by crude extracellular enzymes of T. fusca BD25 resulted in the detection of aromatic compounds at concentrations of 0.6 microg ml(-1) and 8.7 microg ml(-1), respectively. Hydrolysis of oat spelt xylan by T. fusca BD25 extracellular enzymes yielded a mixture of xylose, xylotriose and putative substituted-xylotriose, while the products of ball-milled wheat straw hydrolysis were xylose, glucose and a small oligomer present in the digest. The results highlight the ability of culture supernatant from T. fusca to release both simple sugars and aromatic compounds from lignocellulosic substrates and suggest a role for this organism in the biobleaching of pulp.     

Aneuploidies, chromosome aberrations and dominant gene mutations detected in 113,913 consecutive newborn children in Mexico

Data on 113,913 liveborn children from a hospital in Guadalajara, Jalisco (Mexico), were analysed for birth defects (BD); mutation rates were calculated for sporadic aneuploidy, chromosome aberrations and dominant gene mutations. The results showed a general incidence of 13.92 BD cases per 1000 liveborns, of which 1.64% were chromosomal abnormalities, 1.50% were aneuploid, 0.14% were structural chromosome aberrations and 3.23% were dominant gene mutations. The mutation rates were 8.20 x 10(-4) chromosomal abnormalities, 7.5 x 10(-4) aneuploidies, 7.0 x 10(-5) chromosome aberrations and 1.61 x 10(-3) dominant gene mutations/gamete/generation, respectively. The lethality rate was 15.32% of the liveborns with BD. The described findings estimate the incidence of new human mutants detected at birth in a sample of the Mexican population. They show that the rate for some aneuploidies are similar to those found in other populations previously reported in the literature but the rates of chromosome and dominant gene mutations were different.     

Errors in estimating lung liquid volume in fetal lambs when using radiolabeled serum albumin and blue dextran.

Fetal lung liquid volume is usually determined by using radio-iodinated serum albumin (RISA) or blue dextran (BD) as volume tracers. We tested the reliability of both tracers at 124 (G124) and 142 days of gestation (G142; term = G147) when the labels were employed simultaneously. We measured the proportion of label bound reversibly to the lung, or apparently lost from the lung compartment, by washing out the lung with saline and 5% albumin. At G124, volume estimates with the two labels were similar. At G142, the volume estimate with BD (36.3 +/- 8.7 ml/kg of body wt) was higher (P < 0. 05) than with RISA (22.3 +/- 3.5 ml/kg). This difference resulted from reversible binding of BD, because 5% albumin washout released 38.5 +/- 4.0% of the BD added at the start of the experiment but a lesser amount of RISA (9.8 +/- 0.7%; P < 0.05). At G142, when RISA was used alone, its reversible binding was 1.3 +/- 0.2%. Background absorbance increased during experiments, giving rise to an apparent increase in BD concentration. We conclude that RISA is an effective tracer for lung liquid volume determination in the fetal lamb, whereas our findings of substantial epithelial binding of BD and large changes in background absorbance demonstrate that, under the conditions of our experiments, BD is a poor tracer close to term.