Glycoengineered factor IX variants with improved pharmacokinetics and subcutaneous efficacy.

BACKGROUND: The rapid clearance of factor IX (FIX) necessitates frequent intravenous administration to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration would be a preferred route of administration but is limited by bioavailability. OBJECTIVES: To improve the pharmacokinetics (PK) and bioavailability of FIX, a screen was performed to identify positions for the introduction of novel glycosylation sites with maximal effect on PK and maintenance of coagulation activity. METHODS: Two hundred fifty-one variants, each containing one additional N-linked glycosylation site, were screened in vitro, and the PK profiles of selected variants mapping to spatially distinct regions of FIX were evaluated in mice. Optimal variants were combined, and their PK and efficacy were determined in mice with hemophilia B. RESULTS: Variants that mapped to spatially distinct regions of the FIX structure exhibited different degrees of improved PK and enabled selection of optimized sites while minimizing the loss of FIX activity. Combining the most effective N-glycan sites in the same FIX molecule resulted in further improvements in PK. An optimized variant containing three novel N-glycan sites (at amino acids 103, 151, and 228), and the activity enhancing 338A variant had double the specific activity of wild-type FIX, exhibited 4.5-fold reduced clearance and 2.4-fold increased subcutaneous bioavailability, and was efficacious at a fivefold lower mass dose than wild-type FIX after subcutaneous injection in a bleeding model in mice with hemophilia B. CONCLUSIONS: Glycoengineering was used to significantly improve the subcutaneous PK and efficacy of FIX and may have advantages for subcutaneous dosing.

von Willebrand factor contributes to longer half-life of PEGylated factor VIII in vivo.

PEGylation of B-domain deleted factor VIII (PEG-FVIII-BDD) prolongs the half-life of the molecule by approximately twofold in animals (Mei et al., Blood 2010; 116: 270). To investigate the role of von Willebrand factor (vWF) in the catabolism of PEG-FVIII-BDD in vivo, a FVIII-BDD mutant (F8V), which is incapable of binding vWF, was generated by deleting the vWF-binding region in the a3 domain of FVIIII-BDD. F8V was expressed, purified and PEGylated by site-specific conjugation. The biochemical and biological properties of F8V and PEGylated F8V (PEG-F8V) were evaluated in vitro and in vivo. The specific activity of purified F8V by a chromogenic assay was similar to FVIII-BDD and PEGylation had minimal impact on the specific activity of F8V in this assay. Analysis by Biacore indicated that both F8V and PEG-F8V display greatly reduced vWF binding in vitro. Pharmacokinetic studies in FVIII knockout (HaemA) mice showed that the terminal half-life (T1/2 ) of F8V was dramatically reduced relative to FVIII-BDD (0.6 h vs. 6.03 h). PEGylation of F8V promoted a significant increase in T1/2 , although PEGylation did not fully compensate for the loss in vWF binding. PEG-F8V showed a shorter T1/2 than PEG-FVIII-BDD both in HaemA mice (7.7 h vs. 14.3 h) and in Sprague-Dawley male rats (2.0 ± 0.3 h vs. 6.0 ± 0.5 h). These data demonstrated that vWF contributes to the longer T1/2 of PEG-FVIII-BDD. Furthermore, this suggests that the clearance of the FVIII:vWF complex, through vWF receptors, is not the sole factor which places an upper limit on the duration of PEG-FVIII circulation in plasma.

Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K.

We have shown previously that the AKT2 pathway is essential for cell survival and important in malignant transformation. In this study, we demonstrate elevated kinase levels of AKT2 and phosphatidylinositol-3-OH kinase (PI3K) in 32 of 80 primary breast carcinomas. The majority of the cases with the activation are estrogen receptor alpha (ERalpha) positive, which prompted us to examine whether AKT2 regulates ERalpha activity. We found that constitutively activated AKT2 or AKT2 activated by epidermal growth factor or insulin-like growth factor-1 promotes the transcriptional activity of ERalpha. This effect occurred in the absence or presence of estrogen. Activated AKT2 phosphorylates ERalpha in vitro and in vivo, but it does not phosphorylate a mutant ERalpha in which ser-167 was replaced by Ala. The PI3K inhibitor, wortmannin, abolishes both the phosphorylation and transcriptional activity of ERalpha induced by AKT2. However, AKT2-induced ERalpha activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, implicating that AKT2-activated ERalpha contributes to tamoxifen resistance. Moreover, we found that ERalpha binds to the p85alpha regulatory subunit of PI3K in the absence or presence of estradiol in epithelial cells and subsequently activates PI3K/AKT2, suggesting ERalpha regulation of PI3K/AKT2 through a nontranscriptional and ligand-independent mechanism. These data indicate that regulation between the ERalpha and PI3K/AKT2 pathway (ERalpha-PI3K/AKT2-ERalpha) may play an important role in pathogenesis of human breast cancer and could contribute to ligand-independent breast cancer cell growth.

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

Identification of AKT-regulated genes in inducible MERAkt cells.

AKT/protein kinase B plays a critical role in the phosphoinositide 3-kinase (PI3-kinase) pathway regulating cell growth, differentiation, and oncogenic transformation. Akt1-regulated genes were identified by cDNA array hybridization analysis using an inducible AKT1 protein, MERAKT. Treatment of MERAkt cells with estrogen receptor ligands resulted in phosphorylative activation of MERAKT. Genes differentially expressed in MERAkt/NIH3T3 cells treated with tamoxifen, raloxifene, ICI-182780, and ZK955, were identified at 3 and 20 h. AKT activation resulted in the repression of c-myc, early growth response 1 (EGR1), transforming growth factor beta receptor III (TGF-betar III), and thrombospondin-1 (THBS1). Although c-myc induction is often associated with oncogenic transformation, the c-myc repression observed here is consistent with the anti-apoptotic function of AKT. Repression of THBS1 and EGR1 is consistent with the known pro-angiogenic functions of AKT. AKT-regulated genes were found to be largely distinct from platelet-derived growth factor-beta (PDGFbeta)-regulated genes; only T-cell death-associated gene 51 (TDAG51) was induced in both cases. In contrast to their repression by AKT, c-myc, THBS1, and EGR1 were induced by PDGFbeta, indicating negative interference between elements upstream and downstream of AKT1 in the PDGFbeta signal transduction pathway.

Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.

We previously demonstrated that AKT2, a member of protein kinase B family, is activated by a number of growth factors via Ras and PI 3-kinase signaling pathways. Here, we report the frequent activation of AKT2 in human primary ovarian cancer and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase (PI 3-kinase)/Akt pathway. In vitro AKT2 kinase assay analyses in 91 ovarian cancer specimens revealed elevated levels of AKT2 activity (>3-fold) in 33 cases (36.3%). The majority of tumors displaying activated AKT2 were high grade and stages III and IV. Immunostaining and Western blot analyses using a phospho-ser-473 Akt antibody that detects the activated form of AKT2 (AKT2 phosphorylated at serine-474) confirmed the frequent activation of AKT2 in ovarian cancer specimens. Phosphorylated AKT2 in tumor specimens localized to the cell membrane and cytoplasm but not the nucleus. To address the mechanism of AKT2 activation, we measured in vitro PI 3-kinase activity in 43 ovarian cancer specimens, including the 33 cases displaying elevated AKT2 activation. High levels of PI 3-kinase activity were observed in 20 cases, 15 of which also exhibited AKT2 activation. The remaining five cases displayed elevated AKT1 activation. Among the cases with elevated AKT2, but not PI 3-kinase activity (18 cases), three showed down-regulation of PTEN protein expression. Inhibition of PI 3-kinase/AKT2 by wortmannin or LY294002 induces apoptosis in ovarian cancer cells exhibiting activation of the PI 3-kinase/AKT2 pathway. These findings demonstrate for the first time that activation of AKT2 is a common occurrence in human ovarian cancer and that PI 3-kinase/Akt pathway may be an important target for ovarian cancer intervention.

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

In situ hybridization for gastrin-releasing peptide receptor (GRP receptor) expression in prostatic carcinoma.

Bombesin-like peptides (BLPs), which have been implicated in the regulation of growth of prostatic carcinoma cells, are a product of neuroendocrine cells frequently found in prostate tissue and are postulated to play a role in the initiation or progression of prostatic carcinoma. In this report, we examined the expression, in human prostate tissue, of mRNA encoding the 3 known receptors that respond to BLPs in humans, i.e., gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor and bombesin receptor subtype 3 (BRS-3). Competitive rt-PCR experiments demonstrated the widespread but variable expression of GRP receptor mRNA in fresh-frozen specimens of prostatic carcinoma (12 cases) and benign prostatic hypertrophy (6 cases). NMB receptor mRNA expression was also widespread, but its level was less variable than GRP receptor message. In contrast, we could not detect BRS-3 mRNA in most tissue samples by rt-PCR. To address which cells in the prostate express the GRP receptor, we used in situ hybridization methods to stain selectively GRP receptor mRNA. GRP receptor mRNA was expressed predominantly in the luminal and basal epithelial cells in both histologically normal and cancerous glands within sections of normal (3 cases) and diseased (37 cases) tissue. GRP receptor mRNA staining in cancerous tissue ranged widely from very intense to not detectable (about 30% of the cases), while normal tissue consistently displayed a low level of message staining. Taken together, our results demonstrate expression of the GRP receptor in a high percentage of basal and/or luminal epithelial cells of normal and diseased prostate tissues.

Bombesin-like peptide receptor subtypes promote mitogenesis, which requires persistent receptor signaling.

Bombesin-like peptides (BLPs) can regulate the growth of normal and transformed cells. To compare the relative activities of the three known human BLP receptor subtypes [i.e., the gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor, or BLP receptor subtype 3] in growth regulation, we expressed each receptor in a receptor-deficient host, Balb/3T3 cells. None of the receptor agonists used in our study promoted DNA synthesis by quiescent parental, nontransfected Balb/3T3 cells. Using clones stably transfected with the NMB receptor however, we found that NMB stimulated the incorporation of [3H]thymidine 2.5- to 8-fold over basal levels. The greatest net stimulation of [3H]thymidine incorporation occurred when the medium contained insulin. In quiescent Balb/3T3 cells transfected with the GRP receptor, GRP promoted a 15-fold increase in DNA synthesis in the absence of insulin or other growth factors. GRP also induced the labeling of a large percentage (53%) of the cells with bromodeoxyuridine. To determine the length of time that GRP receptor signaling was required to drive quiescent cells into the S phase of the cell cycle, we blocked GRP receptor signaling by addition of a competitive GRP receptor antagonist at different times after stimulating cells with GRP. Our data demonstrate that persistent GRP receptor signaling throughout a large part of the G1 phase of the cell cycle is important in the mitogenic effects of GRP in these cells. Hitherto uncharacterized GRP receptor signaling pathways may be important in this process. BLPs also stimulated a mitogenic response by transfectants expressing the BLP receptor subtype 3 if insulin was contained in the medium. Taken together, these studies indicate that all three BLP receptor subtypes may contribute to growth regulation in vivo.

Discovery of high affinity bombesin receptor subtype 3 agonists.

Human bombesin receptor subtype 3 (BRS-3) was cloned based on its homology to the human gastrin-releasing peptide (GRP) receptor and neuromedin B (NMB) receptor. Some bombesin-like peptides were shown to activate BRS-3 expressed in Xenopus laevis oocytes, but only at relatively high concentrations, which suggests that BRS-3 is an orphan receptor. To study the pharmacology of BRS-3 in the context of a mammalian cell, we used BR2 cells, which are Balb/3T3 fibroblasts transfected with BRS-3 cDNA. A number of bombesin-like peptides found in mammals and amphibians stimulated calcium mobilization in BR2 cells but exhibited no effect on nontransfected parental Balb/3T3 cells. Of these peptides, NMB (EC50 approximately 1-10 microM) was the most active for stimulation of calcium mobilization. Testing of a series of NMB analogs truncated at the amino terminus and carboxyl terminus indicated that the minimal size of NMB required for retention of full activity was Ac-NMB(3-10). Systematically replacing each residue with alanine, or changing its chirality, demonstrated that the carboxyl-terminal residues His8, Phe9, and Met10 of NMB are important for optimal activity. We also tested whether a number of bombesin (BN) analogs that are potent pure or partial antagonists of the GRP receptor can activate BRS-3 in BR2 cells. One such analog, D-Phe6-BN(6-13) propyl amide, activated BRS-3-mediated calcium mobilization with an EC50 level of 84 nM. Through additional synthesis, we generated a significantly more potent analog, D-Phe6-Phe13-BN(6-13) propyl amide, which displayed an EC50 level of 5 nM for activation of BRS-3. Taken together, our data show that the core portions of bombesin-like peptides required for activation of BRS-3 are similar to those necessary for activation of the GRP and NMB receptors and thus provide pharmacological evidence that BRS-3 is in the BN receptor family. Furthermore, we have identified an agonist of BRS-3, namely D-Phe6-Phe13-BN(6-13) propyl amide, which is roughly 1000-fold more potent than BRS-3 agonists described previously.

Gastrin-releasing peptide receptor signaling resulting in growth inhibition.

We demonstrate that gastrin-releasing peptide (GRP) can inhibit the proliferation of human immortal nontumorigenic (184-B5) mammary epithelial cells ectopically expressing the human GRP receptor. Growth of Balb 3T3 cells ectopically expressing relatively high levels of the GRP receptor was also inhibited by GRP; however, growth of transfectants expressing lower levels of the receptor was not inhibited. Compared with Balb 3T3 cells, mammary epithelial cells could be rendered sensitive to growth inhibition by GRP by the expression of fewer GRP receptors. GRP also stimulated DNA synthesis in quiescent, serum-starved Balb 3T3 transfectants. In clones that were sensitive to growth inhibition by GRP by virtue of their expression of relatively high levels of the GRP receptor, the dose-response curve of GRP-stimulated DNA synthesis was bell shaped. This is consistent with our conclusion that the growth-inhibiting activity of GRP required the activation of a relatively large pool of receptors in Balb 3T3 cells. Significantly, prostaglandin H synthase inhibitors, which block the production of prostaglandins from arachidonic acid, reduced GRP-inhibitory effects on DNA synthesis. We also compared a number of GRP-stimulated signaling pathways in Balb 3T3 clones that were sensitive or insensitive to growth inhibition by GRP, including cAMP formation, phospholipase C activation, calcium mobilization, and arachidonic acid formation. Taken together, these results demonstrate a novel GRP receptor-coupled signal pathway promoting growth inhibition in which prostaglandin H synthase plays a significant role.

Differential activation of human gastrin-releasing peptide receptor-mediated responses by bombesin analogs.

To enable the detailed pharmacological characterization of five bombesin (BN) analogs with respect to the human gastrin-releasing peptide (GRP) receptor, we ectopically expressed the receptor in BALB/3T3 cells. In such cells (termed GR1 cells), GRP stimulated DNA synthesis and Ca2+ mobilization. Two of these analogs, D-Phe6-BN(6-13) methyl ester (Ki = 1.38 +/- 0.07 nM) and 4-pyridyl-CO-His7-D-Ala11-Lys12-COCH2CH2-phenyl- BN(7-13) methyl amide (Ki = 2.17 +/- 0.05 nM), were pure antagonists of GRP-stimulated DNA synthesis in GR1 cells (IC50 = 14 +/- 8.5 nM and 5.1 +/- 2.0 nM, respectively), whereas three analogs, Leu13-psi-Leu14-BN (Ki = 21.6 +/- 2.2 nM), D-Phe6-BN(6-13) ethyl amide (Ki = 5.17 +/- 0.64 nM), and D-Phe6-BN(6-13) propyl amide (Ki = 0.68 +/- 0.01 nM), displayed significant partial agonistic activity. Although three analogs promoted mitogenesis in GR1 cells, none of the analogs stimulated calcium mobilization in GR1 cells. This dichotomy was not limited to transfected cells, because the same result was obtained for D-Phe6-BN(6-13) propyl amide using human fetal lung cells, which naturally express the GRP receptor. We also assessed the effect of BN analogs on calcium mobilization in transfected GR9 cells expressing about 30 times higher levels of the GRP receptor, compared with GR1 cells. D-Phe6-BN(6-13) ethyl amide, D-Phe6-BN(6-13) propyl amide, and Leu13-psi-Leu14-BN were partial agonists of the intracellular Ca2+ mobilization response of GR9 cells. One conclusion consistent with our data is that GRP-stimulated DNA synthesis requires the activation of far fewer receptors than does GRP-stimulated calcium mobilization. Thus, analogs with a small amount of agonist activity can trigger a mitogenic response but not an intracellular Ca2+ mobilization response, unless cells express a high level of receptors. These studies also provide evidence that the promotion of DNA synthesis in quiescent GR1 or human fetal lung cells via the GRP receptor does not require mobilization of intracellular Ca2+.

Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.

Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and was purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose and ligand affinity chromatography. The purified receptor displayed a single diffuse band with a Mr of 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment of the receptor with N-glycanase, removing N-linked oligosaccharide moieties, the protein yielded a Mr = 38,000 band. These results agree with the Mr value estimated for the GRP receptor that was labeled on Swiss 3T3 cells by cross-linking to 125I-GRP1-27. GRP1-27 bound to the purified receptor with a Kd of 0.038 +/- 0.019 nM. By comparison, the soluble receptor in unfractionated extracts and intact membranes displayed a Kd for GRP1-27 of 0.036 +/- 0.003 nM and 0.13 +/- 0.04 nM, respectively. The relative potencies of a series of GRP analogs for the soluble receptor and intact membranes indicated that the extraction procedure did not significantly alter the receptor's ligand binding specificity. However coupling of the receptor to its guanyl nucleotide regulatory protein was not maintained in the soluble extract, and a G-protein did not co-purify with the receptor. Physiological concentrations of NaCl greatly inhibited the binding of some GRP analogs to the receptor, while the binding of other analogs was not affected. A domain on the GRP molecule involving Lys-13 or Arg-17 was identified which promoted binding to the GRP receptor under conditions of low ionic strength. These findings aided the development of an effective ligand affinity resin for the purification of the GRP receptor.

Tracheotomies: a 10-year experience in 319 children.

A retrospective analysis was made of 319 patients having tracheotomies at the Hospital for Sick Children in Toronto between 1976 and 1985. In comparison to a similar study at the same institution completed 15 years earlier, the average number of tracheotomies per year has declined by half, because almost no tracheotomies are now done in patients with epiglottitis and tracheitis. Tracheotomies for children with CNS disorders and craniofacial anomalies are relatively more frequent. The average duration of tracheotomy is almost 1 year (339 days). Complications occurred in 30% of patients, but tracheotomy-related mortality was less than 1%.

Aberrant responses to growth-regulatory signals by variant kidney epithelial cells.

Cultures that achieved a higher cell density than expected were noted during study of growth regulation in monkey kidney epithelial cells of the BSC-1 line. Multiplication of the variant cells was accelerated, compared with parental cells, as the cultures approached confluence. Cytogenetic analysis, immunofluorescence antibody reactions with specific monkey serum, isoenzyme analysis, microbiological studies, and lack of growth in soft agar indicated that the variant cells were not a contaminating cell type, lacked new isoenzymes, were free of microbial contamination, and were not transformed. Confluent variant cultures did not respond to a purified growth inhibitor protein produced by BSC-1 cells that inhibits multiplication and reduces cell Na content in subconfluent variant and parental cells. Vasopressin, which is a mitogen for parental cells, was a potent growth inhibitor for confluent cultures of variant cells. Low-K or high-Na media, which stimulate proliferation of parental cells, had no effect on growth of the variant cell line. These results suggest that enhanced multiplication of the variant cells is mediated by altered signal transduction pathways and/or receptors for growth-regulatory molecules.

Otoplasty: an update.

Otoplasty is a common cosmetic surgical procedure. Various techniques have been described through the years, many of these addressing only specific aspects of the deformity rather than overall correction of the multiple defects that often occur. To achieve optimal results, an understanding of the embryology of the external ear and an appreciation of the development of otoplasty techniques are useful. Current surgical concepts are emphasized and the authors' graduated approach to otoplasty is presented. Thirty-five cases over a four-year period are reviewed. All achieved a satisfactory cosmetic result; there were 5 complications, 3 being due to Mustarde suture pull-away. Two of these required revision, and revision was also required in another patient who developed a hypertrophic scar. The remaining complication was a minor suture granuloma.

Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum.

Yeast secretory mutant sec53 cells accumulate inactive secretory glycoprotein precursors that remain associated with the endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). The possibility that precursor polypeptides fail to penetrate completely into the ER lumen was tested by examining the protease accessibility of accumulated invertase, mating pheromone precursor prepro-alpha-factor and the vacuolar protein precursor procarboxypeptidase Y in cell lysates. In all three cases, the secretory protein precursors are protected from the action of exogenous protease unless the membrane is permeabilized by including Triton X-100 or saponin in the incubation. These results suggest that the sec53 defect allows complete polypeptide translocation. Consistent with this interpretation, the precursor of invertase accumulates in a signal peptide-processed form. In addition, invertase and prepro-alpha-factor precursors contain a small amount of possibly aberrant carbohydrate. In mutant cells or in wild type cells treated with tunicamycin, a 10-kDa fragment of the N terminus of mature invertase assumes a conformation that is resistant to trypsin with or without detergent. This domain may be associated with an ER protein or may simply assume an unusual conformation as a consequence of deficient glycosyl modification.