©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Quantitative Polymerase Chain Reaction of Lysyl Oxidase mRNA in Malignantly Transformed Human Cell Lines Demonstrates That Their Low Lysyl Oxidase Activity Is Due to Low Quantities of Its mRNA and Low Levels of Transcription of the Respective Gene (*)

(Received for publication, April 25, 1995; and in revised form, June 23, 1995)

Eija-Riitta Hämäläinen (1) Ritva Kemppainen (1) Helena Kuivaniemi (2) Gerard Tromp (2) Antti Vaheri (3) Taina Pihlajaniemi (1) Kari I. Kivirikko (1)(§)

From the  (1)Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland, the (2)Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, and the (3)Department of Virology, University of Helsinki, FIN-00018 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lysyl oxidase (EC 1.4.3.13), an extracellular copper amino oxidase, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the -amino group in certain lysine and hydroxylysine residues. We developed here a polymerase chain reaction (PCR) method for the quantification of lysyl oxidase mRNA in which a synthetic RNA is used as an internal standard for coamplification with the targeted mRNA. The amount of lysyl oxidase mRNA when studied by Northern blot analysis and the number of lysyl oxidase mRNA molecules when determined by the quantitative PCR method were found to be markedly low in various malignantly transformed cell lines relative to control cell lines, quantitative PCR indicating values of about 2-10% of those in the controls. No difference was found in the number of beta-actin mRNA molecules between the transformed cells and the controls. Nuclear runoff experiments indicated that most if not all of the decrease in the number of lysyl oxidase mRNA molecules can be explained by diminished transcription of the respective gene.


INTRODUCTION

Lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the -amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin (for reviews, see (1) and (2) ). Molecular cloning and complete cDNA-derived amino acid sequences have been reported for the rat (3, 4) , human(5, 6) , and chick (7) enzymes, which were found to be synthesized in precursor forms of 411, 417, and 420 amino acids, respectively. The human lysyl oxidase gene is located on chromosome 5 (5, 6) and the mouse gene on chromosome 18(8, 9, 10) , both genes consisting of seven exons(11, 12) . Increased lysyl oxidase activity has been reported in fibrotic disorders(1) , while a deficiency is found in two X-linked, recessively inherited human disorders, the occipital horn syndrome and Menkes syndrome, and in the X-linked recessively inherited mottled series of allelic mutant mice(13, 14, 15) . In these X-linked disorders, the low enzyme activity appears to be secondary to abnormalities in copper metabolism.

Lysyl oxidase activity is markedly low in the culture medium of many malignantly transformed human cell lines(16) . The cDNA-derived amino acid sequence of the mouse ras recision gene, rrg(17) , has been found to match that of rat lysyl oxidase (18) , suggesting that rrg and lysyl oxidase are identical. The levels of rrg mRNA (17) and lysyl oxidase activity (18) are markedly decreased in NIH 3T3 cells transformed by LTR-c-Ha-ras compared with those in nontransformed NIH 3T3 or in cells after reversion following prolonged treatment with interferon-beta(17) . Transfection of the revertants with antisense rrg constructs leads to a transformed morphology again, and the cells become tumorigenic in nude mice(17) .

The purpose of this work was to explore further the reasons for the low lysyl oxidase activity observed in the culture medium of malignantly transformed cells. For this purpose, we developed a PCR (^1)method for the quantification of lysyl oxidase mRNA in which a synthetic RNA (cRNA) is used as an internal standard for coamplification with the target mRNA. We also studied the amount of lysyl oxidase mRNA by Northern blotting and the production of this mRNA in in vitro nuclear runoff experiments.


MATERIALS AND METHODS

Cell Culture

The cultured human cell lines were embryonal skin (HES) and lung (HEL, WI-38) fibroblasts and adult skin (20009, 9505, 9011) fibroblasts, SV40 virus-transformed WI-38 cells (VA-13), fibrosarcoma cells (HT-1080), embryonal rhabdomyosarcoma cells (RD), choriocarcinoma cells (JEG-3), and melanoma cells (G-361). The cells were cultured at 37 °C at 5% CO(2) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum, 100 units/ml penicillin and streptomycin.

RNA Preparations and Northern Hybridization

Total cytoplasmic RNA was isolated from cultured cells by acid guanidinium thiocyanate-phenol-chloroform extraction (19) and stored dry at -70 °C until used. The RNA yield was measured by absorbance at 260 nm. Electrophoresis of total RNA was performed on a 1.0% agarose gel containing 2 M formaldehyde, and RNA was transferred to a nitrocellulose filter and hybridized with P-labeled HLO20 cDNA for human lysyl oxidase (5) and glyceraldehyde 3-phosphate dehydrogenase(20) .

Assay for Lysyl Oxidase Activity

Lysyl oxidase activity was assayed from the conditioned medium. A tritiated, insoluble elastin substrate was prepared from the aortae of 16-day-old chick embryos, which had been pulsed in organ culture with L-4,5-[^3H]lysine in the presence of beta-aminopropionitrile(21) .

Internal and External Controls

The lysyl oxidase and beta-actin constructs were made by PCR using cDNA clones as templates. Two constructs were made for lysyl oxidase. The first, used to quantify the mRNA, was from the 3`-region of lysyl oxidase cDNA, and the second, from the region of the first exon, was used to quantify nuclear RNA by means of runoff transcription reactions. Primers 1 (sense) and 2 (antisense) were designed so that they contained EcoRI sites in the 5`-end. Primers 3 (antisense) and 4 (sense) contained HindIII sites. The sequences of the primers (Table 1) used for quantification of the lysyl oxidase mRNA were so distinct from those of the recently described human lysyl oxidase-like gene (22) that no amplification of the latter sequences can take place. Two amplification reactions were performed, one with primers 1 and 3 and the other with primers 2 and 4. Next, the amplified products, containing restriction sites for EcoRI and HindIII, were digested with HindIII and ligated. The ligated fragment was used as a template for the third PCR with primers 1 and 2. This fragment had EcoRI sites at both ends and contained a deletion.



The cDNA for the proalpha1 chain of type III procollagen(23, 24) contained an internal TaqI restriction site in the amplified region and was thus digested with TaqI, religated, and the deleted PCR fragment amplified with primers 1 and 2. All the PCR products with deletions were cloned into Bluescript SK, and plasmid isolation was performed by the CsCl method(25) . The constructs were sequenced to verify their identity and orientation.

In Vitro RNA and cDNA Synthesis

Deleted constructs were linearized and used in an in vitro transcription reaction according to the protocol of Ambion. The synthetic RNA products (cRNA) were run on PAGE to verify their size and integrity. Two methods were initially used to determine the amount of cRNA. First, 5 µCi of [P]UTP (specific activity, 400Ci/mmol; Amersham) was added to the in vitro transcription reaction, and incorporation was determined by trichloroacetic acid precipitation as advised by Ambion. Second, the absorbances of the cRNA samples were measured at 260 nm. cDNA was synthesized with 0.5-10 µg of total cellular RNA and 10^7-10^9 molecules of cRNA, 1 times RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl(2)), 0.5 mM dNTP, 5 mM dithiothreitol, random hexamer primers, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) in a total volume of 20 µl and incubated at 37 °C for 60 min.

Nuclear Runoff Transcription

The nuclear runoff transcriptions were carried out according to Greenberg and Ziff(26) . The amount of nuclear RNA was determined by absorbance at 260 nm, and this RNA was used for cDNA synthesis with the cRNA made for sequences encoding the first exon of the lysyl oxidase gene.

PCR

PCR was performed at a final concentration of 1 times PCR buffer, 0.16 mM of each dNTP, 50 pmol of downstream primer, 50 pmol of upstream primer P end-labeled with [-P]ATP and T4-polynucleotide kinase, and 1 unit of thermostable DNA polymerase (Dynazyme) in a total volume of 50 µl.

Each PCR cycle in the lysyl oxidase and type III procollagen proalpha1 chain amplification reactions included 90 s of denaturation at 94 °C, 80 s of annealing at 60 °C, and 90 s of extension at 72 °C. The conditions for beta-actin were the same, except that the annealing temperature was 58 °C.

Quantitative Analysis

Preliminary PCRs were performed to establish the number of control molecules necessary to obtain signals of approximately equal intensities from the endogenous cDNA and control cRNA molecules. Serial dilutions of the appropriate ratios of cRNA to cDNA were subsequently prepared and amplified. 10 µl of each PCR product was separated on 6% PAGE, dried, and autoradiographed. The bands corresponding to each specific PCR product were then excised from the dried gels, and the amount of radioactivity incorporated was determined by liquid scintillation counting. The cRNA incorporation data for each experiment were plotted, and the linear portion of the curve was identified. Linear regression with a y intercept of zero was performed on the data that were linear. The slope of the regression line was used to compute the numbers of endogenous molecules per picogram of total cellular RNA. A few experiments were deemed inconclusive since the correlation of the control data with the regression line was poor (R^2 < 0.75). For the dilutions over which the control data were linear, the computed number of endogenous molecules usually also demonstrated linearity.


RESULTS

Northern Blot Analysis

The amount of lysyl oxidase mRNA in five malignantly transformed human cell lines and six controls was determined by Northern blotting. HLO20, a cDNA probe for lysyl oxidase(5) , hybridizes to multiple mRNAs, the sizes being 5.5, 4.3, 2.4, and 2.0 kilobases. The 4.3-kilobase band, corresponding to the major species, was detected in RNA from all the control cell lines, i.e. embryonal lung (WI-38 and HEL) and skin fibroblasts (HES) and adult skin fibroblasts(9011) (Fig. 1). No signal for lysyl oxidase mRNA was seen in RNA from the transformed cell lines, including SV40-transformed WI-38 cells (VA-13), melanoma cells (G-361), fibrosarcoma cells (HT-1080), choriocarcinoma cells (JEG-3), and embryonal rhabdomyosarcoma cells (RD). When the same blot was hybridized with a cDNA probe for glyceraldehyde 3-phosphate dehydrogenase, all the RNA samples yielded a band of the expected size and of about equal intensity. Since the RNA appeared to be intact, the failure to see any band with the lysyl oxidase probe must have been due to a marked reduction in the amount of the lysyl oxidase mRNA.


Figure 1: Northern blot analysis of lysyl oxidase mRNA in malignantly transformed and control cell lines. Total cytoplasmic RNA, 20 µg, was hybridized to a P-labeled cDNA probe, HLO20(5) , for human lysyl oxidase. The malignantly transformed cell lines are SV40 virus transformed WI-38 cells (VA-13), melanoma cells (G-361), fibrosarcoma cells (HT-1080), choriocarcinoma cells (JEG-3), and embryonal rhabdomyosarcoma cells (RD). The control cell lines are embryonal lung (WI-38, HEL) and skin (HES) fibroblasts and adult fibroblasts(9011). The 4.3-kilobase band corresponding to the major species of the human lysyl oxidase mRNA is shown.



Quantitative PCR for Lysyl Oxidase mRNA with Internal Standard

To quantify the lysyl oxidase mRNA in the transformed cell lines, a PCR-based assay was developed. An internal standard containing a deletion of 60 bp in the middle of the lysyl oxidase cDNA was constructed. In vitro transcribed RNA (cRNA) was generated from this control construct. Known amounts of the cRNA were mixed together with known amounts of total RNA isolated from cultured cells, and cDNA synthesis, PCR, and PAGE analysis were carried out. The PCR products obtained with the cDNAs derived from the cRNA and the cellular RNA differed in size by 60 bp and were therefore easy to distinguish on PAGE. Fig. 2shows an example of the PCR products specific to lysyl oxidase mRNA and the cRNA. In this experiment, the cDNAs were synthesized under standard conditions with 0.93 µg of total cellular RNA from the control cell line 9011 and 10.96 µg of total cellular RNA from the fibrosarcoma cell line HT-1080. In both cases, the RNA samples also contained 8.44 times 10^7 molecules of the lysyl oxidase cRNA as an internal standard. Serial 1:2 dilutions were made, and PCR amplifications were carried out. The internal standard had two functions. First, it served as an internal control for the reverse transcription and PCR amplification reactions, and second, it was used to generate a standard curve for quantifying the target mRNA in experimental samples. Since the same primers were used in the PCR for both templates, there were no primer efficiency differences between the standard and the target RNAs. Different dilutions of cDNA synthesis mixtures containing both the target mRNA and standard cRNA were coamplified in the same tube in the exponential phase, and the amount of target mRNA was determined by interpolating against the cRNA standard curve. The amplification efficiencies were determined as described by Wang et al.(27) . The efficiency was 40-46% (n = 6) for cDNA derived from RNA isolated from three cell lines and 41-47% (n = 6) for cDNA derived from the internal standard. The corresponding values for beta-actin mRNA were 32-40% and 35-41%.


Figure 2: Autoradiogram of lysyl oxidase mRNA (568 bp) and the internal standard cRNA (508 bp)-specific PCR products from one control(9011) and one transformed (HT-1080) cell line. 0.93 µg of total cellular RNA from the 9011 cells and 10.96 µg of RNA from the HT-1080 cells were reverse transcribed in the presence of 8.44 times 10^7 molecules of the internal standard cRNA. Serial 1:2 dilutions (from right to left) were performed, the PCR amplifications were carried out, and the products were separated on 6% PAGE, dried, and autoradiographed.



Quantities of Lysyl Oxidase mRNA in Malignantly Transformed and Control Cell Lines

As previously reported (16) , the lysyl oxidase activity of all the transformed cell lines was very low, being below the limit of accurate measurement (Table 2). The number of lysyl oxidase mRNA molecules per picogram of total cellular RNA varied between 340 and 440 in four control cell lines, but the numbers in the transformed cell lines were only about 2-10% of these (p < 0.0005), being 7-40 molecules/pg (Table 2). No differences in the number of beta-actin mRNA molecules were found between the transformed cells and the controls (Table 2). The number of mRNA molecules for the proalpha1 chain of type III procollagen had decreased, although to a smaller extent than for lysyl oxidase; the values in the four control cell lines ranged from 380 to 980 molecules/pg RNA, whereas that value in the VA-13 cells was 72 molecules and that in the RD cells was 126 molecules/pg RNA (details not shown).



Nuclear Runoff Transcription

The levels of transcription of the lysyl oxidase gene were measured in different cell lines using nuclear runoff assays. Quantification of pre-mRNA molecules in such experiments demonstrated that the level of transcription of the lysyl oxidase gene is markedly lower (p < 0.0005) in transformed cells than in the controls (Table 3). The decrease in the transcription level was not quite as marked, however, as the decrease in the amounts of lysyl oxidase mRNA molecules and enzyme activity.




DISCUSSION

The quantitative PCR method developed here for human lysyl oxidase mRNA follows the principles described by Wang et al.(27) . The specific mRNA and an internal standard cRNA containing a 60-bp deletion are coamplified in the same reaction and with the same primers, and the PCR products are separated by PAGE and quantified. The numbers of endogenous mRNA molecules can then be computed from the curve determined for the internal standard.

The data obtained with the quantitative PCR indicated that the number of lysyl oxidase mRNA molecules per picogram of total RNA was about 340-440 in the four control cell lines, the mean being about 8% of the mean for the values of about 2000-6600 molecules/pg determined for the mRNA molecules that encode beta-actin and about 60% of the mean for the values of 380-980 molecules/pg for those encoding the proalpha1 chain of type III procollagen. The last-mentioned value does not indicate a ratio of the number of mRNA molecules for lysyl oxidase to the number for its polypeptide substrate because type III procollagen represents only about 15-20% of the total collagen synthesized by cultured skin fibroblasts (see (28) and references therein). Thus, the number of lysyl oxidase mRNA molecules may be about one-tenth of the number for its polypeptide substrates.

The number of lysyl oxidase mRNA molecules in malignantly transformed cell lines was very low, both when measured by Northern blotting and when assayed with the quantitative PCR. The latter indicated values of about 2-10% of those in the controls. Lysyl oxidase activity in malignantly transformed cell lines has previously been reported as about 10% that in controls(16) . The present assays indicated an even lower percentage, but as the activity levels in the transformed cells were below the limit of accurate measurement both in the previous study (16) and here, the actual levels probably do not differ greatly. The magnitude of the decrease in the number of lysyl oxidase mRNA molecules thus appears to be very similar to that in enzyme activity, indicating that the low enzyme activity is due to a pretranslational mechanism.

Nuclear runoff experiments indicated that most if not all of the decrease in the lysyl oxidase mRNA levels can be explained by diminished transcription of the respective gene. However, as the runoff values for the transformed cells were not quite as low as those for the mRNA molecules by comparison with the control cells, the data do not exclude the possibility that decreased stability of the lysyl oxidase mRNA may have contributed to the low mRNA and enzyme activity levels.

The present data obtained with several human tumor cell lines are in good agreement with those reported for the rrg mRNA, which appears to be identical to the lysyl oxidase mRNA in NIH 3T3 cells transformed by LTR-c-Ha-ras (see Introduction). Thus, the findings reported for the LTR-cHa-ras-transformed NIH 3T3 cells appear to be similar in a number of tumor cell types. It is of particular interest that the data obtained with the NIH 3T3 cells suggest that the lysyl oxidase gene may have a tumor suppressor activity(17, 18) . However, the mechanisms by which this gene may achieve this activity are currently unknown. It is likewise unknown what cis-acting elements in the lysyl oxidase gene and trans-acting factors in the cells are responsible for the low lysyl oxidase mRNA levels in the tumor cells. The promoter region and downstream sequences in the lysyl oxidase gene contain a number of potential binding sites for various transcription factors (12) , but the elements involved in the decrease in the transcription of the gene in transformed cells remain to be identified.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of the Academy of Finland, the Sigrid Jusélius Foundation, and the Paulo Foundation. 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: Dept. of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland. Tel.: 358-81-5375801; Fax: 358-81-5375810.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).


ACKNOWLEDGEMENTS

We are grateful to Riitta Polojärvi and Aira Harju for technical assistance.


REFERENCES

  1. Kagan, H. M. (1986) in Biology of Extracellular Matrix (Mecham, R. P., ed) Vol. 1, pp. 321-398, Academic Press, Orlando, FL
  2. Kagan, H. M., and Trackman, P. C. (1991) Am. J. Respir. Cell Mol. Biol. 5,206-210 [Medline] [Order article via Infotrieve]
  3. Trackman, P. C., Pratt, A. M., Wolanski, A., Tang, S. S., Offner, G. D., Troxler, R. F., and Kagan, H. M. (1990) Biochemistry 29,4863-4870 [Medline] [Order article via Infotrieve]
  4. Trackman, P. C., Pratt, A. M., Wolanski, A, Tang, S. S., Offner, G. D., Troxler, R. F., and Kagan, H. M. (1991) Biochemistry 30,8282 [Medline] [Order article via Infotrieve]
  5. Hämäläinen, E.-R., Jones, T., Sheer, D., Taskinen, K., Pihlajaniemi, T., and Kivirikko, K. I. (1991) Genomics 11,508-516 [Medline] [Order article via Infotrieve]
  6. Mariani, T. J., Trackman, P. C., Kagan, H. M., Eddy, R. L., Shows, T. B., Boyd, C. D., and Deak, S. B. (1992) Matrix 12,242-248 [Medline] [Order article via Infotrieve]
  7. Wu, Y., Rich, C. B., Lincecum, J., Trackman, P. C., Kagan, H. M., and Foster, J. A. (1992) J. Biol. Chem. 267,24199-24206 [Abstract/Free Full Text]
  8. Mock, B. A., Contente, S., Kenyon, K., Friedman, R. M., and Kozak, C. A. (1992) Genomics 14,822-823 [Medline] [Order article via Infotrieve]
  9. Chang, Y. S., Svinarich, D. M., Yang, T. P., and Krawetz, S. A. (1993) Cytogenet. Cell Genet. 63,47-49 [Medline] [Order article via Infotrieve]
  10. Lossie, A. C., Buckwalter, M. S., and Camper, S. A. (1993) Mamm. Genome 4,177-178 [Medline] [Order article via Infotrieve]
  11. Czsisar, K., Mariani, T. J., Gosin, J. S., Deak, S. B., and Boyd, C. D. (1993) Genomics 16,401-406 [CrossRef][Medline] [Order article via Infotrieve]
  12. Hämäläinen, E.-R., Kemppainen, R., Pihlajaniemi, T., and Kivirikko, K. I. (1993) Genomics 17,544-548 [CrossRef][Medline] [Order article via Infotrieve]
  13. Kivirikko, K. I., and Kuivaniemi, H. (1987) in Connective Tissue Disease (Uitto, J., and Peredja, A. J., eds) pp. 263-292, Dekker, New York
  14. Danks, D. M. (1992) in Connective Tissue and Its Heritable Diseases: Molecular, Genetic and Medical Aspects (Royce, P. M., and Steinmann, B., eds) pp. 487-506, Wiley-Liss, New York
  15. Kivirikko, K. I. (1993) Ann. Med. 25,113-126 [Medline] [Order article via Infotrieve]
  16. Kuivaniemi, H., Korhonen, R.-M., Vaheri, A., and Kivirikko, K. I. (1986) FEBS Lett. 195,261-264 [CrossRef][Medline] [Order article via Infotrieve]
  17. Contente, S., Kenyon, K., Rimoldi, D., and Friedman, R. M. (1990) Science 249,796-798 [Medline] [Order article via Infotrieve]
  18. Kenyon, K., Contente, S., Trackman, P. C., Tang, J., Kagan, H. M., and Friedman, R. M. (1991) Science 253,802 [Medline] [Order article via Infotrieve]
  19. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159 [CrossRef][Medline] [Order article via Infotrieve]
  20. Arcari, P., Martinelli, R., and Salvatore, F. (1984) Nucleic Acids Res. 12,9179-9189 [Abstract]
  21. Kagan, H. M., and Sullivan, K. A. (1982) Methods Enzymol. 82,637-650 [Medline] [Order article via Infotrieve]
  22. Kenyon, K., Modi, W. S., Contente, S., and Friedman, R. M. (1993) J. Biol. Chem. 268,18435-18437 [Abstract/Free Full Text]
  23. Ala-Kokko, L., Kontusaari, S., Baldwin, C. T., Kuivaniemi, H., and Prockop, D. J. (1989) Biochem. J. 260,509-516 [Medline] [Order article via Infotrieve]
  24. Tromp, G., Kuivaniemi, H., Stolle C., Pope, F. M., and Prockop, D. J. (1989) J. Biol. Chem. 264,19313-19317 [Abstract/Free Full Text]
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Greenberg, M. E., and Ziff, E. B. (1984) Nature 311,433-438 [Medline] [Order article via Infotrieve]
  27. Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,9717-9721 [Abstract]
  28. Prockop, D. J., and Kivirikko, K. I. (1995) Annu. Rev. Biochem. 64,403-434 [CrossRef][Medline] [Order article via Infotrieve]

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