©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier (*)

Manuel M. Sánchez del Pino (§) , Richard A. Hawkins , Darryl R. Peterson

From the (1)Department of Physiology and Biophysics, Finch University of Health Sciences, The Chicago Medical School, North Chicago, Illinois 60064

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that -glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5`-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Na/K-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na/K-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.


INTRODUCTION

Isolated membrane vesicles provide a convenient tool to study the function and characteristics of membranes at a molecular level(1, 2) . Ideally, a pure and homogeneous membrane vesicle population is desired. However, this is not possible in most cases, and a mixture of membrane vesicles from different plasma membrane domains is commonly obtained. The purity or origin of the membranes is usually assessed by measuring the relative specific activity (RSA)()of marker enzymes, which identify specific membrane populations. Some problems are associated with this practice. First, starting with a tissue containing several cell types, with which the activity of the final preparation is compared, may produce a misleading high value of RSA. Second, if the protein density of a particular membrane domain is higher than the others, the RSA may be lower, although the same enrichment is achieved. Thus, only limited quantitative conclusions can be obtained.

A variety of proteins has been associated with the blood-brain barrier (BBB)(3, 4, 5) , including antigens of unknown function, receptors, and enzymes. However, it is possible that no molecular components exist that are exclusively located in BBB-specific endothelial cells(5) . Traditionally, in the absence of completely specific markers, the membrane-bound enzymes -glutamyl transpeptidase (GGT), alkaline phosphatase, 5`-nucleotidase, and Na/K-ATPase are the most commonly used markers. All studies have shown alkaline phosphatase on the luminal membrane of brain endothelial cells, most of them exclusively. Some reports indicate, however, that alkaline phosphatase is on both membranes (reviewed in Ref. 3). GGT is probably the most widely used marker for the BBB(5) ; yet, there are only two cytochemical studies showing GGT localization at the electron microscopic level. The first indicates that the enzyme is exclusively on the luminal membrane(6) , whereas the second ascribes an entirely abluminal position(7) . Biochemical results (8) suggest that alkaline phosphatase and GGT are located on both luminal and abluminal sides of the brain endothelial cells. GGT and alkaline phosphatase colocalize in the luminal membrane of epithelial cells, and, in fact, the expression of both enzymes parallel the appearance of epithelial characteristics in tissue culture(9) . If brain endothelial cells can be considered as a specialized endothelium, with epithelial properties mediating the transport of substrates between blood and brain(10) , one might expect the same distribution observed in epithelial cells.

Cytochemical studies located Na/K-ATPase exclusively (8) or primarily (11) on the abluminal membrane. However, a more recent report claims that enzyme activity is equally distributed on both membranes(12) . Vorbrodt (3) noted that when the incubation time with the Na/K-ATPase substrate is increased, some reaction product appears on the luminal side as well as the abluminal surface, which suggests heterogeneity in Na/K-ATPase activity. A multiplicity of isoforms (3 and 2 subunits of the Na/K-ATPase) has been shown recently using Western blot analysis(13) , indicating that six different isoenzymes may be expressed in brain endothelial cells. Collectively, these results suggest that while abluminal membranes contain most of the Na/K-ATPase activity, or the most active isoform, some activity may also be present in luminal membranes.

We have previously shown that membrane vesicles isolated from bovine brain endothelial cells can be used to study transport(14) . These results were interpreted according to the consensus opinion (3) that GGT and alkaline phosphatase were located on the luminal membrane and Na/K-ATPase was on the abluminal membrane. However, as indicated above, contradictory results concerning the location of markers have been reported that may affect the interpretation of transport experiments. The following experiments were designed to show that the marker enzyme information can be analyzed in such a way that their location on each membrane domain can be deduced. Furthermore, the relative composition of luminal and abluminal membranes in each fraction can be determined, and the contribution of each membrane to the measured transport may be quantified.


EXPERIMENTAL PROCEDURES

Materials

N-(Methylamino)-[1-C]isobutyric acid (48.4-56.3 mCi/mmol) and [G-H]ouabain (15.4 Ci/mmol) were purchased from DuPont NEN. Collagenase type IA and cytochrome c type IV were obtained from Sigma. Bio-Rad protein assay was purchased from Bio-Rad.

Isolation of Endothelial Cell Membranes

Brain microvessels were isolated essentially as described by Pardridge et al. (15) with only minor modifications(14) . Brain endothelial cell membranes were isolated by a modification (14) of the procedure described by Betz et al.(8) . Briefly, stored microvessels were incubated at 37 °C for 25 min in isolation buffer (101 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2HO, 1.2 mM KHPO, 1.2 mM MgSO, and 14.5 mM HEPES, pH 7.4) containing 1800 units of collagenase type IA per gram of capillary. The collagenase-treated capillaries were homogenized in TSEM buffer (250 mM sucrose, 0.1 mM EGTA, 0.5 mM MgCl, and 10 mM Tris-HCl, pH 7.4) using a Tissuemizer (Tekmar, Cincinnati, OH), and the cellular debris was removed by centrifugation. To the membrane fraction contained in the supernatant, solid MgSO was added to a final concentration of 10 mM. The suspension was centrifuged at 3,200 g for 15 min at 4 °C, and the supernatant was centrifuged at 90,000 g for 1 h at 4 °C in a Ty 35 Beckman rotor. The pellet was resuspended in TSEM and layered on top of a discontinuous Ficoll gradient (5, 10, 15, and 20% Ficoll). After centrifuging at 162,500 g for 2.5 h at 4 °C in a 70 Ti Beckman rotor, five fractions corresponding to the interfaces between the different Ficoll layers and the pellet were collected. All fractions were diluted in storage buffer (290 mM mannitol and 10 mM HEPES-Tris, pH 7.4) and left overnight in an ice bath. Samples were centrifuged at 90,000 g for 30 min at 4 °C in a Ty 35 Beckman rotor, and the pellet was resuspended in 2-3 ml of the same buffer. Aliquots were stored at -80 °C.

Marker Enzymes

GGT, alkaline phosphatase, 5`-nucleotidase, and Na/K-ATPase activities were used as plasma membrane markers(3) . Amino acid transport system A was also used as a plasma membrane marker. GGT and alkaline phosphatase activities were measured in the presence of 0.1% (v/v) Triton X-100 (16). 5`-Nucleotidase activity was determined in the presence of 0.02% (v/v) Triton X-100(17) . The Na/K-ATPase activity was determined by measuring the K-dependent, ouabain-sensitive phosphatase activity (17) or the binding of radiolabeled ouabain(14) . The activity of amino acid transport system A was determined by measuring the initial rate of uptake of 100 µM [C]MeAIB in the presence of NaCl or KCl as indicated in the accompanying paper(18) . The specific activity was calculated by subtracting the activity in the presence of KCl from the activity in the presence of NaCl and expressed as pmol MeAIB min mg of protein. N-Acetyl--D-glucosaminidase activity was used as a lysosomal marker and measured in the presence of 0.2% (v/v) Triton X-100(19) . The activity of cytochrome c-oxidase, a mitochondrial enzyme, was measured in the presence of 0.1% (v/v) Triton X-100(20) .

Electron Microscopy

Samples of isolated capillaries were critical point dried after dehydration. They were then positioned on an aluminum stub and sputter coated with gold. The specimen was examined in a JEOL scanning electron microscope (JSM-35) at 20 kV.

Isolated membranes were also prepared for transmission electron microscopy. Samples of the Ficoll gradient fractions were centrifuged at 37,500 g for 25 min at 4 °C, and the pelleted membranes were fixed in modified Tyrode's solution(21) , post-fixed in osmium tetroxide, dehydrated in an ethanol series, cleared in propylene oxide, and embedded in Embed 812 (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were stained with lead citrate and uranyl acetate and examined under a Zeiss EM 10C electron microscope.

Morphological analysis of isolated membrane vesicles was performed on micrographs of the luminal and abluminal membrane-rich fractions using the procedure described by Weibel and Bolender(22) .

Protein Determination

Protein concentration was determined using the Bio-Rad protein microassay, with bovine serum albumin as the standard, based on the method of Bradford(23) .

Analysis of Marker Enzyme Data

The procedure is based on two assumptions. First, the markers are located exclusively on the plasma membrane, either luminal or abluminal, which is the case for those used (reviewed in Ref. 3). Second, contamination by cells other than capillary endothelial cells is negligible. The second assumption is supported by the results described below. To know the content of luminal and abluminal membranes in each fraction and in which membrane the marker enzymes are located, the following analysis was used.

The activity of each plasma membrane marker was normalized by the activity present in the whole gradient and expressed as a percentage,

On-line formulae not verified for accuracy

where M is the percentage of marker m in fraction i, and U is the activity units of marker m in fraction i. The array of markers (a-m) in each fraction (1-i) was obtained as indicated in . Similarly, the amount of luminal and abluminal membrane in each fraction was expressed as a percentage of the total amount contained in the whole gradient. The content of luminal and abluminal membranes in the gradient can be considered a good representation of the total plasma membrane population since similar recoveries of the plasma membrane markers were obtained at each step during the isolation procedure. Thus, the membrane preparation that we loaded onto the Ficoll gradient contained the same proportion of the plasma membrane markers as the initial homogenate.

The fraction of plasma membrane marker activity located on the luminal membrane, f, is the activity of that marker located on the luminal membrane divided by the total activity present in both luminal and abluminal membranes. This fraction is 0 when all the activity is located on the abluminal membrane and 1 when all the activity is located on the luminal membrane. The fraction of marker activity located on the abluminal membrane is 1 - f.

The percentage of marker present in each fraction is given as follows,

On-line formulae not verified for accuracy

where L and A are the percentages of luminal and abluminal membrane in fraction i, respectively. The array of calculated marker activities is obtained from the definitions shown in . The percentage of marker in each fraction was determined experimentally. Since the luminal (L) and abluminal (A) content was expressed as percentages, the values in one of the fractions was a combination of the values in the other four fractions. The number of unknown parameters were the luminal (L) and abluminal (A) content in only four fractions and the f values for the markers. To determine these parameters, Equation 2 was used to find the best least squares fit to the measured amount of markers in each fraction.

The fitting was carried out using the spreadsheet Microsoft Excel as follows. An initial set of f values was provided to start the fitting procedure. The L and A values were calculated as the regression coefficients of f and (1 - f), respectively, in a multiple linear regression analysis between rows in the observed matrix and the f and (1 - f) values, using a built-in function of the program. The measured M values were compared with those obtained using Equation 2 with a first set of estimated parameters. The agreement between measured and calculated results was given by least squares. An iterative procedure (quasi-Newton method) was used to find the f values that produced the best agreement with the observed results. The L and A values were updated after each iteration. The standard errors of the estimated values of L and A were obtained from the multiple linear regression analysis indicated above. At the end of the fitting procedure, a multiple linear regression analysis between columns of the observed matrix and the L and A values was performed. This analysis provided the standard errors of f and (1 - f), the regression coefficients of L and A, respectively.

With the information provided by the method, specifically the luminal and abluminal percentages, the f values for any activity associated with the plasma membrane can be readily determined. To establish the location of a particular marker, its activity in terms of total activity units in only two fractions is needed, as indicated below.

It is clear from Equation 1 that the ratio of a marker activity in two fractions can be obtained using either the percentage of activity (Equation 1) or the total activity. If we calculate this ratio in terms of total activity and combine with Equation 2, we have the following expression,

On-line formulae not verified for accuracy

where U is the total units, L and A are the percentages of luminal and abluminal membranes, respectively, contained in each fraction, and i and j refer to two different fractions. If we now solve for f,

On-line formulae not verified for accuracy

and define R as follows,

On-line formulae not verified for accuracy

then the following equation is obtained:

On-line formulae not verified for accuracy


RESULTS

Isolation of Brain Microvessels

The isolated microvessel preparation appeared to consist, at the light microscope level, of branching capillary segments with only occasional small arterioles and venules and without contamination by other cell types. Scanning electron microscopy showed that the isolated microvessels were devoid of any nerve or glial cells (Fig. 1a). The average diameter of the isolated microvessels was 3.5 ± 0.6 µm (±S.D.), in agreement with the size observed in other capillary beds (24). The absence of blood cell contamination was supported by the results shown in the accompanying paper (18) where no ASC transport system activity present in red blood cells was detected. Thus, the protocol to isolate brain microvessels produced a nearly pure capillary preparation in which endothelial cells constituted the predominant cell type.


Figure 1: a, scanning electron micrograph of isolated brain microvessels. b and c, phase-contrast micrographs of isolated brain capillaries. b, before collagenase treatment, some rounded particles (arrowheads) can be seen at the external surface of capillaries. c, after collagenase treatment and washing of the capillaries, almost no such particles are present at the capillary walls. The bars are 10 µm in length.



Collagenase Treatment

Before collagenase digestion, some rounded bodies were observed (Fig. 1b) scattered along the isolated capillaries. Their location at the outer surface of capillaries suggested that these bodies were pericytes or fragments of glial endfeet. Collagenase treatment, which digested the basement membrane, released them. After centrifugation and resuspension of the collagenase-digested capillaries, none of these particles were seen (Fig. 1c). It is interesting to note that if the observed structures were indeed pericytes, the described protocol might be useful to isolate pericytes associated with the blood-brain barrier.

Isolation of Membrane Vesicles

As we previously showed, the membrane preparations consist of sealed vesicles without detectable contamination by other organelles(14) . Morphological analysis (22) of transmission electron micrographs (not shown) indicated that the average radius of the vesicles was 164 ± 15 (± S.D.) and 125 ± 12 nm for luminal and abluminal membrane-rich fractions, respectively.

Marker Enzyme Distribution

When the RSA for each of the plasma membrane marker enzymes was calculated, a maximum enrichment in the 0/5% Ficoll fraction was observed. The enhancement declined in the remaining fractions (I). The achieved enrichments were between 7 and 20, except for the K-dependent, ouabain-sensitive phosphatase activity. However, when the activities of the markers were expressed as percentages, as indicated under ``Experimental Procedures,'' the difference between markers was apparent (Fig. 2). The GGT activity was concentrated in the first fraction, which corresponds to the 0/5% Ficoll interface, whereas the other markers were concentrated in denser fractions (5/10 and 10/15% Ficoll interfaces). As an additional marker, the Na-dependent transport activity of the amino acid analog MeAIB was used. This analog is specifically transported by system A, which was centered in fraction 10/15. There was good correlation between the measured marker activities and those predicted by Equation 2 (Fig. 3), supporting the validity of the estimated parameters.


Figure 2: Distribution of marker enzymes within the gradient. Enzyme activity is expressed as the percentage of the total activity recovered in the gradient. The markers located in only one membrane are indicated by the solidlines. The brokenlines indicate markers present in both membranes. AP, alkaline phosphatase; 5N, 5`-nucleotidase; KP, K-dependent, ouabain-sensitive phosphatase; A, system A transport activity; P+I, pellet plus material between interfaces.




Figure 3: Correlation between measured and predicted marker activity. The marker activity, expressed as the percentage of the total activity recovered in the gradient, was calculated using Equation 2, and the result was plotted as a function of the measured activity. The line is the regression fit, which has a slope of 0.986 and a correlation coefficient of 0.986. AP, alkaline phosphatase; 5N, 5`-nucleotidase; KP, K-dependent, ouabain-sensitive phosphatase; A, system A transport activity.



The results obtained using the mathematical analysis described above indicated that fraction 0/5 was enriched in one membrane domain and fractions 10/15 and 15/20 in the other (Tables IV and V). As discussed below, the low and high density membrane vesicles were considered to be enriched in luminal and abluminal membranes, respectively. Thus, fraction 0/5 and 10/15 can be considered as luminal and abluminal membrane-rich fractions (), respectively, which is a similar distribution to that found by Betz et al.(8) . Although fraction 15/20 has a higher abluminal/luminal ratio than fraction 10/15, the latter was selected as most suitable for transport studies for two reasons. First, the amount of protein obtained in fraction 10/15 is much higher than that in 15/20, allowing many more experiments. Second, contamination by lysosomes and mitochondria is lower in fraction 10/15.

The results of the analysis also indicated that GGT activity is located almost exclusively in the luminal membrane, whereas alkaline phosphatase and 5`-nucleotidase activities are evenly distributed in both luminal and abluminal membranes (). K-dependent, ouabain-sensitive phosphatase activity appeared to be located mainly on the abluminal side, although some activity was present also in the luminal side (about 25%). When ouabain binding was measured, the affinity for ouabain was lower in the luminal than in the abluminal membrane-rich fractions (Fig. 4), indicating that activity may be indeed present in both membranes but due to different isoforms. The transport activity of system A appeared to be located exclusively on the abluminal membrane, which makes it a good marker for abluminal membranes.


Figure 4: Binding of ouabain to luminal and abluminal membrane-rich vesicles. An Eadie-Hofstee plot of the fractional saturation of ouabain binding sites present in luminal and abluminal membrane-rich fractions shows two dissociation constants (± S.E.) for ouabain.




DISCUSSION

A novel analytical approach was used to treat the enzyme marker data and extract quantitative information concerning the contribution of the individual membranes in transport experiments. The analysis of the marker enzymes described here offers some advantages over the more conventional approaches using RSA values. It is not necessary to measure the activity in the initial homogenate, which, due to the high content of cellular debris, may produce inconsistent results. It allows the use of a broader variety of membrane markers, even some with unknown location. Transport activities and receptor binding sites are particularly appropriate markers to use with the described protocol. It represents a quantitative, not qualitative, approach so that once the distribution of the plasma membrane domains (the fraction of luminal and abluminal) is known, the location of any activity can be assigned by measuring the activity in only two fractions, and the exact contribution of each membrane domain to the activity being measured can be determined.

The presence of two membrane vesicle populations (luminal and abluminal) was indicated by the different distribution of plasma membrane markers. One population was enriched in GGT, whereas the other was enriched in Na/K-ATPase and system A activities. The designation of luminal and abluminal has been made on the basis of the following considerations.

1) Na/K-ATPase activity has always been found on the abluminal membrane. In the cases where some activity was also present on the luminal membrane, the same or more activity was on the abluminal side, indicating that this enzyme is, at least primarily, an abluminal membrane-bound enzyme(3, 11, 12) .

2) Na-dependent transport of amino acids has been measured only using isolated brain microvessels where the abluminal membrane is the most accessible membrane, suggesting that this transport system is most probably located on the abluminal side (25, 26). A luminal location of system A would predict a significant transport of small neutral amino acids, the preferred substrates of this system, across the BBB. However, transport of these amino acids is undetectable in experiments in vivo, where substrate is presented to luminal membranes, supporting the abluminal location of this carrier.

3) Na/K-ATPase activity and system A transport activity co-localize primarily to a single membrane population in the current study, implying that both proteins can be considered markers for the same plasma membrane domain ( and Fig. 2). This distribution is opposite that of GGT activity. These observations strengthen the previous conclusions, supporting an abluminal location of both Na/K-ATPase and system A activities.

Although most of the K-dependent, ouabain-sensitive phosphatase activity appeared to be located on the abluminal side, some activity was detected also in the luminal side. The ouabain binding results presented here, along with the results reported by other authors(11, 12) , support the idea that different isoforms are located in each membrane. A multiplicity of isoforms (3 and 2 subunits of the Na/K-ATPase) has been shown recently using Western blot analysis(13) , indicating that as many as six different isoenzymes could be expressed in brain endothelial cells. These results indicate that polarity exists in the sense that abluminal membranes, representing the majority of the activity, contain an isoform with a higher affinity for ouabain, whereas luminal membranes contain a minor component of the activity, which has a lower ouabain affinity. This distribution may favor a net sodium transport from blood to brain, as suggested by some authors (27), because more sodium is removed by the Na/K-ATPase isoform located in abluminal membranes. A polarized distribution of different isoforms of membrane proteins such as glucose transporters has also been shown in other tissues(28) .

GGT activity is located in the luminal membrane. Since the distribution of GGT in the Ficoll gradient is markedly different from that of the Na/K-ATPase and system A activities, which are located on the abluminal membranes, it has been concluded that GGT activity is located on the luminal membrane. This location agrees with the results of Ghandour et al.(6) , identifying this enzyme activity with the luminal membrane of brain endothelial cells.

Alkaline phosphatase and 5`-nucleotidase activities are, on the contrary, equally distributed between luminal and abluminal membranes. The distribution of alkaline phosphatase found here has been reported also by some authors (8) using cytochemical techniques. The finding that 5`-nucleotidase is located on both membranes is, however, in disagreement with some cytochemical studies(3) . A possible explanation for this discrepancy is that, as noted by Vorbrodt(3) , this enzyme is extremely sensitive to the fixatives used to prepare the samples for cytochemical examination. In most cytochemical experiments, the brains are perfused with a fixative, probably creating a concentration gradient of fixative from the luminal to the abluminal sides of the vessels. Thus, it is possible that enzymes located on the luminal membrane are inactivated but those located on the abluminal side are not.

The distribution of membrane markers, schematized in Fig. 5, can be quantitatively determined with the described analysis. The results reported here indicate that luminal and abluminal membranes of brain endothelial cells can be separated and that the properties of each membrane can be studied separately. GGT and amino acid transport system A are suitable markers for luminal and abluminal membranes, respectively. Our results represent a confirmation of the concept, based on indirect evidences(25, 29) , that transport system A is exposed to the brain extracellular fluid. The results also validate the interpretation of our previous results(14) , based on the distribution of GGT and Na/K-ATPase activities, and establish the conditions to further characterize the constituting membranes of the blood-brain barrier.


Figure 5: Schematic representation of the location of plasma membrane markers. The marker distribution is based on data shown in Table V.



  
Table: Array of markers measured in the gradient fractions


  
Table: Array of calculated marker activities


  
Table: Relative specific activity of the marker enzymes in the Ficoll gradient fractions

The protein is expressed in milligrams. The specific activity is expressed in nmol min mg, except for COX, which is A min mg. H, microvessel homogenate; AP, alkaline phosphatase; 5`N, 5`-nucleotidase; KP, K-dependent, ouabain-sensitive phosphatase; COX, cytochrome-c-oxidase; NAG, N-acetyl--D-glucosaminidase, P+I, pellet plus material between interfaces. Specific activity is indicated in parentheses.


  
Table: Distribution of luminal and abluminal membranes in the Ficoll gradient fractions

The luminal and abluminal content is expressed as percentages of the total amount present in the gradient. The luminal and abluminal values represent the average ± S.D. of three isolations. L/A, luminal to abluminal ratio; P+I, pellet plus material between interfaces.


  
Table: Marker enzyme distribution

The f values represent the average ± S.D. of three preparations (alkaline phosphatase and system A measured in two preparations). A membrane preparation is the combination of 4-5 vesicle isolations, each obtained from 8-12 cow brains.



FOOTNOTES

*
This work was supported by Grants NS 16389 and DK42331 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: MRC Unit for Protein Function and Design, Dept. of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.

The abbreviations used are: RSA, relative specific activity; BBB, blood-brain barrier; GGT, -glutamyl transpeptidase.


ACKNOWLEDGEMENTS

We thank Veronika Burmeister for preparing the samples for electron microscopy.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.