©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation (*)

(Received for publication, October 23, 1995; and in revised form, November 15, 1995)

Sachiko Sato (1)(§) Cristina L. Ward (1) Mauri E. Krouse (2) Jeffrey J. Wine (2) Ron R. Kopito (1)(¶)

From the  (1)Department of Biological Sciences and (2)Department of Psychology, Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California 94305-5020

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The common DeltaF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and DeltaF508 in vivo. Incubation of transient and stable DeltaF508 tranfectants with 10% glycerol induced a significant accumulation of DeltaF508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued DeltaF508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) DeltaF508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.


INTRODUCTION

Cystic fibrosis (CF), (^1)a lethal hereditary exocrinopathy affecting approximately one in two thousand live births among populations of Caucasian or northern European descent, is caused by the functional absence of a plasma membrane chloride channel, designated cystic fibrosis transmembrane conductance regulator (CFTR)(1) . The vast majority of severe CF cases in these populations is linked to a single genetic lesion, deletion of a phenylalanine codon (DeltaF508)(2, 3) , which interferes with the folding of newly synthesized CFTR polypeptides. Nascent DeltaF508 molecules fail to traffic to the plasma membrane (4) but rather are retained by the ER quality control mechanism that prevents unfolded or misfolded proteins and unassociated subunits from exiting the ER. Instead, these retained immature DeltaF508 molecules are rapidly degraded (5, 6) in a pre-Golgi compartment by a process that appears to require covalent modification by ubiquitin(7) . Moreover, plasma membrane CFTR-like Cl channel activity can be detected when DeltaF508 is overexpressed (8) or synthesized at reduced temperature (9) , suggesting that Phe does not play an essential role in CFTR function and raising the possibility of therapeutic intervention in CF by increasing the efficiency of DeltaF508 folding.

Glycerol and other polyols are known to stabilize protein conformation (10) , increase the rate of in vitro protein refolding(11) , and increase the kinetics of oligomeric assembly(12) . We report here that treatment of DeltaF508-expressing cells with glycerol dramatically stabilizes newly synthesized DeltaF508 polypeptides and leads to the accumulation, in the plasma membrane, of stable, functional CFTR Cl channels.


EXPERIMENTAL PROCEDURES

Cell Culture and Transient Transfections

Cells were cultured and transfected exactly as described previously(5) . Mutant cDNA constructs were engineered as described in(13) .

Pulse-Chase Experiments and Immunoprecipitation and Immunoblotting

Pulse-chase and immunoblotting experiments were performed as described previously(5) , with the following modifications in the presence of glycerol. Transfected HEK cells were first treated with 5% glycerol in methionine and cysteine-free DMEM supplemented with 5% dialyzed FCS for 30 min. HEK cells, which were floating after this treatment, were spun down and suspended in 10% glycerol in methionine and cysteine-free DMEM-FCS. Cells were pulse-labeled with S-protein labeling mix (1175 Ci/mmol, DuPont NEN) at a concentration of 0.5 mCi/ml for 15 min and chased with DMEM-FCS with 10 mM methionine and 4 mM cysteine.

Kinetics of Glycerol Uptake

Untransfected HEK cells were treated with 7.5% glycerol in DMEM-FCS for 30 min, and cells were spun down and resuspended in 3 ml of DMEM-FCS supplemented with 7.5% glycerol. Glycerol uptake was measured by incubating untransfected HEK cells at 37 °C with [^3H]glycerol (0.1 mCi/ml, DuPont NEN) for various times. Cell-associated [^3H]glycerol was separated from free [^3H]glycerol by rapid centrifugation (25 s at 16,000 times g) through an 80-µl cushion of silicon oil (Versalube, General Electric). After removing the supernatant, the bottom of the tube was cut, and the cell pellets were extracted in 1% Triton X-100 for determination of cell-associated radioactivity. [^3H]Glycerol uptake measurements were adjusted for trapped extracellular volume by using [^14C]sucrose (1.5 µCi/ml) as a fluid-phase marker(14) .

Electrophysiological Recording

After treatment with 10% glycerol for 24 h, glycerol was removed from HEK cells expressing CFTR or DeltaF508 by diluting the glycerol-containing medium slowly with fresh medium over the course of 1 h. After removal of the glycerol, cells were allowed to recover for at least 1 h prior to analysis by whole cell patch clamp recording. The electrodes were made from Dagan LA-16 glass, Sylgard coated and fire polished to give a final resistance of 2-5 megaohms when filled with pipette solution. The bath solution contained in mM: 150 NaCl, 5 KCl, 2.5 CaCl(2), 2.5 MgCl(2), and 10 HEPES buffered to 7.3. The osmolarity was adjusted to 300 mosm/liter. The pipette solution contained in mM: 125 CsCl, 2.5 MgCl(2), 10 EDTA, 3.5 Mg-ATP, 0.5 cAMP, and 10 HEPES buffered to 7.3. The osmolarity was adjusted to 250 mosm/liter to avoid inducing swelling currents. In some experiments 80 units/liter protein kinase A were added to the pipette solution. The experiments were conducted at 22 °C. Whole cell currents were measured using the Axopatch 1-C, and the data were digitized, stored to disc, and analyzed using the program pClamp. The current was filtered at 500 Hz and digitized at 2 KHz.


RESULTS AND DISCUSSION

The effect of glycerol treatment on steady-state expression of DeltaF508 was initially evaluated by immunoblot analysis of detergent extracts of HEK 293 cells transfected with DeltaF508 cDNA (Fig. 1). In untreated cells, only the immature (core glycosylated, 140 kDa) form was detected in cells incubated at 37 °C (Fig. 1A), as previously observed(5) . However, a diffuse immunoreactive band, corresponding to the mobility of mature (complex glycosylated, 165 kDa) CFTR, was apparent in extracts of cells treated with glycerol for 24 h. The steady-state levels of mature DeltaF508 induced by glycerol or by incubation at reduced temperature in these cells were similar, but the effects appear to be additive. The small difference in mobility between mature DeltaF508 rescued by reduced temperature and that rescued by glycerol is probably due to differences in terminal glycosylation(15) . The effect of glycerol on the maturation of DeltaF508 was also observed in a stable line of C127 mammary carcinoma cells expressing DeltaF508 cDNA (16) (Fig. 1B), indicating that this phenomenon is not unique to transiently transfected HEK cells. Glycerol significantly increased expression of mature DeltaF508 in these cells above basal levels and above the levels induced by incubation at 26 °C. The effect on steady-state expression of mature DeltaF508 in C127 cells was maximal at 10% glycerol; concentrations above or below this level did not support DeltaF508 maturation. By contrast, similar levels of DeltaF508 maturation were observed in HEK cells between 8 and 15% (data not shown). Glycerol did not appear to be acutely toxic to either cell type. Cell viability (determined by trypan blue exclusion) after 24-h exposure to 10% glycerol was between 75% in HEK cells and 90% in C127 cells. Mature DeltaF508 accumulated to clearly detectable levels in C127 cells 6 h following the addition of glycerol and continued to accumulate up to 48 h (Fig. 1C). This effect was reversible; the level of mature DeltaF508 in glycerol-treated C127 cells decreased with time following glycerol removal at a rate consistent with the half-life of mature DeltaF508 protein estimated from pulse-chase experiments (see below). Taken together, these data demonstrate that exposure of DeltaF508-expressing cells to 10% glycerol partially rescues the ``misprocessing'' phenotype of the mutant protein.


Figure 1: Effect of glycerol on steady-state expression of DeltaF508. A, HEK cells expressing DeltaF508 cDNA were treated with 15% glycerol at either 26 or 37 °C for 24 h prior to lysing and analyzed by immunoblotting for steady-state levels of mature (m) or immature (i) DeltaF508. B, concentration dependence of glycerol effect in C127 cells. Cells expressing DeltaF508 (lanes 2-6) were incubated for 24 h with media supplemented with the indicated concentrations of glycerol and evaluated by immunoblotting for mature or immature DeltaF508. Wild-type (wt) CFTR expressed in C127 cells (lane 1) is included for reference. In lane 7 (asterisk) cells were incubated in the absence of glycerol for 24 h at 26 °C. C, time course of mature DeltaF508 accumulation in cells incubated in the presence of 10% glycerol.



The effect of glycerol on DeltaF508 processing could not be replicated by incubating the cells with similar concentrations of other structurally related polyols (1,2-propanediol or 1,3-propanediol), perhaps because of lower permeability of cell membranes to these agents. Similarly, dimethyl sulfoxide (2%) did not support DeltaF508 maturation; higher concentrations were not tested because of its toxicity. Glycerol, a small (M(r) = 98) polyhydric alcohol, is highly permeant across the plasma membrane of animal cells(17, 18) . In HEK cells incubated at 7.5% glycerol, [^3H]glycerol equilibrated rapidly (t, 5 min) across the plasma membrane (data not shown). As glycerol is uncharged its distribution across the plasma membrane is independent of membrane potential. Thus, it is likely that intracellular and extracellular glycerol concentrations rapidly equilibrate in HEK cells and that glycerol's effect on DeltaF508 processing is due to its high intracellular concentration.

At least two mechanisms could account for the effect of glycerol on the accumulation of mature DeltaF508. One possibility is that glycerol stabilizes a DeltaF508 folding intermediate, which is normally rapidly diverted to the degradation apparatus. The stabilized folding intermediate would remain competent to fold into a conformation resistant to proteolysis and permissive for maturation beyond the ER. A second possibility is that glycerol acts by increasing the stability of a mature but unstable DeltaF508 polypeptide that has escaped ER retention. To discriminate between these models, the kinetics of DeltaF508 maturation and degradation were evaluated by pulse-chase labeling and immunoprecipitation in DeltaF508-transfected HEK cells (Fig. 2). In control cells not treated with glycerol, label in the band corresponding to immature DeltaF508 decayed rapidly and was nearly undetectable after 6 h of chase (Fig. 2A). The tof this decay was estimated to be 45 min (Fig. 2, A and D), similar to previously reported values(5) . No label was detected at the mobility corresponding to the mature protein. By contrast, in the presence of glycerol, the kinetics of immature CFTR degradation were significantly slowed (t= 87 min); some of this label was clearly chased into mature DeltaF508 (Fig. 2, A and D). The fractional conversion of immature DeltaF508 in glycerol-treated (Fig. 2E) cells ranged between 3 and 8% in separate experiments, which is considerable when compared with the 20-25% efficiency of wild-type CFTR processing. The kinetics of wild-type CFTR degradation were also slowed by glycerol, suggesting that the effect of glycerol is not unique to the folding of DeltaF508 molecules (Fig. 2, C and D). Glycerol had no measurable effect on the stability of mature DeltaF508 or CFTR (Fig. 2, B and E). These data suggest that accumulation of mature DeltaF508 in glycerol-treated cells is not the result of an effect on the mature protein and that glycerol stabilizes the immature form of DeltaF508. However, inhibition of DeltaF508 degradation, either directly with protease inhibitors or indirectly by blocking ubiquitination, also stabilizes the immature form of the protein but, unlike glycerol treatment, does not result in any accumulation of mature forms(7) . This disparity in the fate of stabilized immature DeltaF508 molecules suggests that glycerol maintains immature DeltaF508 in a maturation-competent state, either by inhibiting reactions that are off pathway or by enhancing reactions that are on the folding pathway.


Figure 2: Effect of glycerol on degradation and maturation of CFTR and DeltaF508. HEK cells expressing DeltaF508 (A and B) or CFTR (C) were pulse-labeled and chased in the presence or absence of 10% glycerol for the times indicated. m, mature; i, immature. D, kinetics of immature DeltaF508 (squares) or CFTR (circles) degradation in the presence (closed symbols) or absence (open symbols) of glycerol. Inset, semilogarithmic plot of data in D. E, kinetics of DeltaF508 (squares) or CFTR (circles) maturation in the presence (open symbols) or absence (closed symbols) of glycerol. Data shown are derived from two experiments and are representative of 3-5 independent trials.



To rule out the possibility that the effects of glycerol on DeltaF508 processing and degradation are due to a general disruption of the ER quality control machinery, we examined the effect of glycerol on the maturation and degradation of other CFTR mutants that, like DeltaF508, are unable to escape the ER (Fig. 3). Cells expressing the missense mutants D572A and S1251A and a mutant harboring a deletion of exon 13 (DeltaEX13) were pulse-labeled with [S]Met and chased for 5 h in the presence or absence of glycerol (Fig. 3A). These mutants were synthesized as immature polypeptides that were degraded and, unlike DeltaF508, failed to mature even in the presence of glycerol. Thus, some mutations in all three major cytoplasmic domains of CFTR, including the first and second nucleotide binding domains (D572A and S1251A, respectively) as well as the ``R'' domain, can lead to a glycerol-insensitive ER retention phenotype, suggesting that glycerol treatment does not induce a general suppression of ER retention mechanisms. The efficiency of processing and the ability to be rescued by glycerol are also highly dependent upon the nature of the substituted amino acid in CFTR Lys missense mutants (Fig. 3B). Processing of mutants K464R and K464A was inefficient by comparison with wild type and was enhanced by incubation in the presence of 10% glycerol, even after accounting for the unequal label present in the immature precursor in the presence of glycerol (Fig. 3B). By contrast, no maturation was detectable for the mutant K464W in the presence or absence of glycerol. These data support the argument that glycerol rescue of CFTR maturation is not the result of a general suppression of ER quality control and suggest a correlation between the ``leakiness'' of the mutation and its ability to be remediated by glycerol.


Figure 3: The effect of glycerol on non-DeltaF508 CFTR mutants. HEK cells expressing D572A, DeltaEX13, and S1251 (A) or various substitutions at Lys (B) were pulse-labeled (p) for 15 min and chased for 5 h (c) in the presence or the absence of glycerol. The mobilities of the mature (m) and immature (i) forms of CFTR and immature DeltaEX13 (i*) are indicated for reference.



Although these data establish that glycerol treatment facilitates the maturation of DeltaF508 molecules to a post-ER compartment, they do not establish that the ``rescued'' DeltaF508 molecules actually move to and are functional at the plasma membrane. To test the functional surface expression of glycerol-rescued DeltaF508 molecules, whole cell CFTR currents were examined in HEK cells expressing CFTR or DeltaF508 by the patch-clamp technique (Fig. 4). Large (56 ± 4 pA/picofarad) rapidly activated, cAMP-dependent Cl selective currents were readily observed in cells expressing CFTR but not in control (not glycerol-treated) cells expressing DeltaF508 (1.54 ± 0.13 pA/picofarad). By contrast, significant (13.31 ± 2.3 pA/picofarad; p < 0.002 compared with untreated; Student's t test) Cl currents, although slower activating than wild type, were observed in glycerol-treated cells expressing DeltaF508 cDNA. These currents were not observed in the absence of cAMP, as expected of CFTR. The difference in maximal current level and activation kinetics between CFTR and glycerol-treated DeltaF508 expressing cells is likely due both to the lower steady-state expression of mature DeltaF508 and to the lower open probability of the mutant channels(19) .


Figure 4: Glycerol treatment induces the expression of plasma membrane DeltaF508 CFTR Cl channels in the HEK cells. A, the mean whole cell maximum current density measured at -50 mV. The currents were divided by the cell capacitance in order to compare results from cell to cell. The currents in DeltaF508 cells not exposed to cAMP (n = 8, column 1) or not glycerol-treated (n = 6, column 2) had maximum current at t = 0 after going whole cell. The currents in glycerol-treated DeltaF508 cells exposed to protein kinase A and/or cAMP (n = 9, column 3) increased slowly and peaked at 3 min. CFTR-expressing cells responded rapidly to intracellular cAMP (n = 3, column 4), peaking at less than 1 min. pF, picofarad. B, typical whole cell currents seen in 3 cells exposed to intracellular cAMP. The currents on the left are the currents seen upon break in, and the currents on the right are currents near the peak of the response. The currents shown are in response to voltage steps from +100 mV to -75 mV in steps of -25 mV from a holding potential of -50 mV. The currents showed linear current-voltage (IV) behavior and no time dependence.



Collectively, these data suggest a model in which glycerol, a short chain polyhydric alcohol, serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and its subsequent release from the quality control apparatus. As degradation of immature CFTR and DeltaF508 is initiated without an apparent lag following translation(5) , we propose that glycerol serves to stabilize nascent DeltaF508 chains soon after or during translation. In this role, glycerol would function as a chemical chaperone, much as HSP70 serves as a molecular chaperone. Our data suggest that these effects are not due to a generalized breakdown of ER quality control nor to stabilization of cell surface mature DeltaF508 molecules that have escaped quality control surveillance. We hypothesize that glycerol stabilizes an early intermediate in CFTR folding that lies at a branch point between productive folding (on pathway) and competing non-productive (off pathway) steps. In this respect the effect of glycerol on DeltaF508 is similar to ``osmotic remedial'' mutants previously observed in yeast (20) and Escherichia coli(21) . These mutations are temperature-sensitive and can be reversed by increasing the osmotic potential of the incubation medium causing the microorganisms to synthesize and accumulate high concentrations of intracellular osmolytes such as glycerol(22) . Interestingly, we observe a strong correlation between the temperature sensitivity of CFTR mutations like DeltaF508, K464R, and K464A (data not shown) and their ability to be remediated by glycerol. Glycerol may provide a useful tool to manipulate the temperature-sensitive phenotypes of CFTR and perhaps other genes at non-permissive temperatures.

Our data establish the precedent that both the intracellular processing and the membrane Cl transport phenotypes of the DeltaF508 mutation can be remediated by chemical means. These data should stimulate a search for other small membrane-permeant molecules, which may be more effective or more easily delivered than glycerol at enhancing DeltaF508 processing. Finally, these data may have implications for the study or treatment of other diseases, including Alzheimer's, retinitis pigmentosa, and proteinase inhibitor deficiency that are associated with protein misfolding.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK43994. This work was done during the tenure of an established investigatorship of the American Heart Association (to R. R. K.). 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.

§
Supported by the Cystic Fibrosis Foundation and a Human Frontiers Science Program long term fellowship.

To whom correspondence should be addressed. Tel.: 415-723-7581; Fax: 415-723-8475; kopito@leland.stanford.edu.

(^1)
The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; ER, endoplasmic reticulum.


ACKNOWLEDGEMENTS

We are indebted to William J. Welch (University of California, San Francisco) for stimulating discussions and for suggesting glycerol as a chemical chaperone and to Kevin Gunderson for providing the mutants used in Fig. 3.


REFERENCES

  1. Welsh, M. J., and Smith, A. E. (1993) Cell 73, 1251-1254 [Medline] [Order article via Infotrieve]
  2. Kerem, B., Zielenski, J., Markiewicz, D., Bozon, D., Gazit, E., Yahav, J., Kennedy, D., Riordan, J. R., Collins, F. S., Rommens, J. M., and Tsui, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8447-8451 [Abstract]
  3. Tsui, L.-C. (1992) Trends Genet. 8, 392-398 [Medline] [Order article via Infotrieve]
  4. Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R., and Smith, A. E. (1990) Cell 63, 827-834 [Medline] [Order article via Infotrieve]
  5. Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718 [Abstract/Free Full Text]
  6. Lukacs, G. L., Mohamed, A., Kartner, N., Chang, X.-B., Riordan, J. R., and Grinstein, S. (1994) EMBO J. 13, 6076-6086 [Abstract]
  7. Ward, C. L., and Kopito, R. R. (1995) Cell 83, 121-127 [Medline] [Order article via Infotrieve]
  8. Cheng, S. H., Fang, S. L., Zabner, J., Marshall, J., Piraino, S., Schiavi, S. C., Jefferson, D. M., Welsh, M. J., and Smith, A. E. (1995) Am. J. Physiol. 268, L615-L624
  9. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and Welsh, M. J. (1992) Nature 358, 761-764 [CrossRef][Medline] [Order article via Infotrieve]
  10. Gekko, K., and Timasheff, S. N. (1981) Biochemistry 20, 4667-4676 [Medline] [Order article via Infotrieve]
  11. Sawano, H., Koumoto, Y., Ohta, K., Sasaki, Y., Segawa, S., and Tachibana, H. (1992) FEBS Lett. 303, 11-14 [CrossRef][Medline] [Order article via Infotrieve]
  12. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 765-768 [Abstract]
  13. Gunderson, K. L., and Kopito, R. R. (1995) Cell 82, 231-239 [Medline] [Order article via Infotrieve]
  14. Restrepo, D., Kozody, D. J., Spinelli, L. J., and Knauf, P. A. (1989) Am. J. Physiol. 257, C520-C527
  15. Fukuda, M., Guan, J. L., and Rose, J. K. (1988) J. Biol. Chem. 263, 5314-5318 [Abstract/Free Full Text]
  16. Marshall, J., Fang, S., Ostedgaard, L. S., O'Riordan, C. R., Ferrara, D., Amara, J. F., Hoppe, H., IV, Scheule, R. K., Welsh, M. J., Smith, A. E., and Cheng, S. H. (1994) J. Biol. Chem. 269, 2978-2995
  17. Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwahara, M., Saito, H., Furukawa, T., Nakajima, K., Yamaguchi, Y., and Gojobori, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6269-6273 [Abstract]
  18. Ma, T., Frigeri, A., Hasegawa, H., and Verkman, A. S. (1994) J. Biol. Chem. 269, 21845-21849 [Abstract/Free Full Text]
  19. Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J. P., and Lazdunski, M. (1991) Nature 354, 526-528 [CrossRef][Medline] [Order article via Infotrieve]
  20. Hawthorne, D. C., and Friis, J. (1964) Genetics 50, 829-839 [Free Full Text]
  21. Russell, R. R. (1972) J. Bacteriol. 112, 661-665 [Medline] [Order article via Infotrieve]
  22. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Science 217, 1214-1222 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.