Nancy E. Liyou

Synthesis and properties of crosslinked recombinant pro-resilin

Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage; it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.

Structure of a human carcinogen-converting enzyme, SULT1A1. Structural and kinetic implications of substrate inhibition.

Sulfonation catalyzed by sulfotransferase enzymes plays an important role in chemical defense mechanisms against various xenobiotics but also bioactivates carcinogens. A major human sulfotransferase, SULT1A1, metabolizes and/or bioactivates many endogenous compounds and is implicated in a range of cancers because of its ability to modify diverse promutagen and procarcinogen xenobiotics. The crystal structure of human SULT1A1 reported here is the first sulfotransferase structure complexed with a xenobiotic substrate. An unexpected finding is that the enzyme accommodates not one but two molecules of the xenobiotic model substrate p-nitrophenol in the active site. This result is supported by kinetic data for SULT1A1 that show substrate inhibition for this small xenobiotic. The extended active site of SULT1A1 is consistent with binding of diiodothyronine but cannot easily accommodate β-estradiol, although both are known substrates. This observation, together with evidence for a disorder-order transition in SULT1A1, suggests that the active site is flexible and can adapt its architecture to accept diverse hydrophobic substrates with varying sizes, shapes and flexibility. Thus the crystal structure of SULT1A1 provides the molecular basis for substrate inhibition and reveals the first clues as to how the enzyme sulfonates a wide variety of lipophilic compounds.

A highly elastic tissue sealant based on photopolymerised gelatin

Gelatin is widely used as a medical biomaterial because it is readily available, cheap, biodegradable and demonstrates favourable biocompatibility. Many applications require stabilisation of the biomaterial by chemical crosslinking, and this often involves derivatisation of the protein or treatment with cytotoxic crosslinking agents. We have previously shown that a facile photochemical method, using blue light, a ruthenium catalyst and a persulphate oxidant, produces covalent di-tyrosine crosslinks in resilin and fibrinogen to form stable hydrogel biomaterials. Here we show that various gelatins can also be rapidly crosslinked to form highly elastic (extension to break >650%) and adhesive (stress at break >100 kPa) biomaterials. Although the method does not require derivatisation of the protein, we show that when the phenolic (tyrosine-like) content of gelatin is increased, the crosslinked material becomes resistant to swelling, yet retains considerable elasticity and high adhesive strength. The reagents are not cytotoxic at the concentration used in the photopolymerisation reaction. When tested in vivo in sheep lung, the photopolymerised gelatin effectively sealed a wound in lung tissue from blood and air leakage, was not cytotoxic and did not produce an inflammatory response. The elastic properties, thermal stability, speed of curing and high tissue adhesive strength of this photopolymerised gelatin, offer considerable improvement over current surgical tissue sealants.

The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant.

We recently reported the generation of a highly elastic, crosslinked protein biomaterial via a rapid photochemical process using visible light illumination. In light of these findings, we predicted that other unmodified, tyrosine-rich, self-associating proteins might also be susceptible to this covalent crosslinking method. Here we show that unmodified native fibrinogen can also be photochemically crosslinked into an elastic hydrogel biomaterial through the rapid formation of intermolecular dityrosine. Photochemically crosslinked fibrinogen forms tissue sealant bonds at least 5-fold stronger than commercial fibrin glue and is capable of producing maximum bond strength within 20s. In vitro studies showed that components of the photochemical crosslinking reaction are non-toxic to cells. This material will find useful application in various surgical procedures where rapid curing for high strength tissue sealing is required.

Localization of a Brain Sulfotransferase, SULT4A1, in the Human and Rat Brain: An Immunohistochemical Study

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.

Association of the SULT1A1 R213H polymorphism with colorectal cancer

1. Sulphotransferases are a superfamily of enzymes involved in both detoxification and bioactivation of endogenous and exogenous compounds. The arylsulphotransferase SULT1A1 has been implicated in a decreased activity and thermostability when the wild‐type arginine at position 213 of the coding sequence is substituted by a histidine. SULT1A1 is the isoform primarily associated with the conversion of dietary N‐OH arylamines to DNA binding adducts and is therefore of interest to determine whether this polymorphism is linked to colorectal cancer.

Coronary artery disease is not associated with the E298 → D variant of the constitutive, endothelial nitric oxide synthase gene

To the Editor: Coronary artery disease (CAD) is multifactorial, occurring as a result of polygenic determinants coupled with a number of environmental factors (1). Endothelial nitric oxide is derived from Larginine by the nitric oxide synthase isoform expressed constitutively in endothelial cells (eNOS). In animal models, inhibition of NOS leads to induction of hypertension, abnormal vasoconstriction and tissue hypoxia (2). phenotypes that are among those observed in certain patients with CAD. A strong association has been demonstrated between a point mutation (G8944T) in exon 7 of the eNOS gene (chromosome 12) and CAD (3). This mutation causes substitution of a Glu -, Asp (E294D) at the protein level. The E294D mutation studied falls well outside the functional domains that have been postulated within the eNOS gene sequence (4), so it is thought most likely that the G894T polymorphism represents a marker mutation.

THE A1166C MUTATION IN THE ANGIOTENSIN II TYPE I RECEPTOR AND HYPERTENSION IN THE ELDERLY

1. Using a nested case‐control study of 661 non‐institutionalized elderly (≥ 60 years) residents of Dubbo, New South Wales, Australia, the aim of this study is to determine whether the A1166C polymorphism of the angiotensin II type I (AT1) receptor gene is associated with hypertension in the elderly.

Evaluation of photo‐crosslinked fibrinogen as a rapid and strong tissue adhesive

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Molecular cloning and expression in Escherichia coli of an aquaporin‐like gene from adult buffalo fly (Haematobia irritans exigua)

A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family. This PCR product was labelled with digoxigenin‐dUTP and used as a probe to screen a λgt‐11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da. Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (drip) of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full‐length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap‐sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full‐length coding sequence of AqpBF1 was cloned into the (His)6‐fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)6‐fusion protein was localized predominantly in the membrane fraction of E. coli. The protein was solubilized from E. coli membranes with n‐octyl β‐d‐glucopyranoside and purified by affinity chromatography on a Ni++–sepharose column in the presence of detergent.