Restoring Carboxylates on Highly Modified Alginates Improves Gelation, Tissue Retention and Systemic Capture.

Alginate hydrogels are gaining traction for use in drug delivery, regenerative medicine, and as tissue engineered scaffolds due to their physiological gelation conditions, high tissue biocompatibility, and wide chemical versatility. Traditionally, alginate is decorated at the carboxyl group to carry drug payloads, peptides, or proteins. While low degrees of substitution do not cause noticeable mechanical changes, high degrees of substitution can cause significant losses to alginate properties including complete loss of calcium cross-linking. While most modifications used to decorate alginate deplete the carboxyl groups, we propose that alginate modifications that replenish the carboxyl groups could overcome the loss in gel integrity and mechanics. In this report, we demonstrate that restoring carboxyl groups during functionalization maintains calcium cross-links as well as hydrogel shear-thinning and self-healing properties. In addition, we demonstrate that alginate hydrogels modified to a high degree with azide modifications that restore the carboxyl groups have improved tissue retention at intramuscular injection sites and capture blood-circulating cyclooctynes better than alginate hydrogels modified with azide modifications that deplete the carboxyl groups. Taken together, alginate modifications that restore carboxyl groups could significantly improve alginate hydrogel mechanics for clinical applications. STATEMENT OF SIGNIFICANCE: Chemical modification of hydrogels provides a powerful tool to regulate cellular adhesion, immune response, and biocompatibility with local tissues. Alginate, due to its biocompatibility and easy chemical modification, is being explored for tissue engineering and drug delivery. Unfortunately, modifying alginate to a high degree of substitution consumes carboxyl group, which are necessary for ionic gelation, leading to poor hydrogel crosslinking. We introduce alginate modifications that restore the alginate's carboxyl groups. We demonstrate that modifications that reintroduce carboxyl groups restore gelation and improve gel mechanics and tissue retention. In addition to contributing to a basic science understanding of hydrogel properties, we anticipate our approach will be useful to create tissue engineered scaffolds and drug delivery platforms.

Genetic Determinants of Salmonella Resistance to the Biofilm-Inhibitory Effects of a Synthetic 4-Oxazolidinone Analog.

Biofilms formed by Salmonella enterica are a frequent source of food supply contamination. Since biofilms are inherently resistant to disinfection, new agents capable of preventing biofilm formation are needed. Synthetic analogs of 4-oxazolidinone containing natural products have shown promise as antibiofilm compounds against Gram-positive bacteria. The purpose of our study was 2-fold: to establish the antibiofilm effects and mechanism of action of a synthetic 4-oxazolidinone analog (JJM-ox-3-70) and to establish mechanisms of resistance to this compound in Salmonella enterica serovar Typhimurium (S Typhimurium). JJM-ox-3-70 inhibited biofilm formation but had no effect on cell growth. The antibiofilm effects were linked to disruption of curli fimbriae and flagellar gene expression and alteration in swimming motility, suggesting an effect on multiple cellular processes. Using a 2-step screening approach of defined multigene and single-gene deletion mutant libraries, we identified 3 mutants that produced less biofilm in the presence of JJM-ox-3-70 than the isogenic WT, with phenotypes reversed by complementation in trans Genes responsible for S Typhimurium resistance to the compound included acrB, a component of the major drug efflux pump AcrAB-TolC, and two genes of unknown function (STM0437 and STM1292). The results of this study suggest that JJM-ox-3-70 inhibits biofilm formation by indirect inhibition of extracellular matrix production that may be linked to disruption of flagellar motility. Further work is needed to establish the role of the newly characterized genes as potential mechanisms of biofilm intrinsic antimicrobial resistance.IMPORTANCE Biofilms are resistant to killing by disinfectants and antimicrobials. S. enterica biofilms facilitate long-term host colonization and persistence in food processing environments. Synthetic analogs of 4-oxazolidinone natural products show promise as antibiofilm agents. Here, we show that a synthetic 4-oxazolidinone analog inhibits Salmonella biofilm through effects on both motility and biofilm matrix gene expression. Furthermore, we identify three genes that promote Salmonella resistance to the antibiofilm effects of the compound. This work provides insight into the mechanism of antibiofilm effects of a synthetic 4-oxazolidinone analog in Gram-negative bacteria and demonstrates new mechanisms of intrinsic antimicrobial resistance in Salmonella biofilms.

Examining ubiquitinated peptide enrichment efficiency through an epitope labeled protein.

Ubiquitination is a dynamic process that is responsible for regulation of cellular responses to stimuli in a number of biological systems. Previous efforts to study this post-translational modification have focused on protein enrichment; however, recent research utilizes the presence of the di-glycine (Gly-Gly) remnants following trypsin digestion to immuno-enrich ubiquitinated peptides. Monoclonal antibodies developed to the cleaved ubiquitin modification epitope, (tert-butoxycarbonyl) glycylglycine (Boc-Gly-Gly-NHS)(1), are used to identify the Gly-Gly signature. Here, we have successfully generated the Boc-Gly-Gly-NHS modification and showed that when conjugated to a lysine containing protein, such as lysozyme, it can be applied as a standard protein to examine ubiquitinated peptide enrichment within a complex background.

Penetrating ulcer of the thoracic aorta: what is it? How do we recognize it? How do we manage it?

BACKGROUND: Although classic type A and B aortic dissections have been well described, less is known about the natural history of penetrating atherosclerotic ulcers of the thoracic aorta. This study differentiates penetrating ulcer from aortic dissection, determines the clinical features and natural history of these ulcers, and establishes appropriate correlates regarding optimal treatment. METHODS: A retrospective review of patient records and imaging studies was conducted with 198 patients with initial diagnoses of aortic dissection (86 type A, 112 type B) at our institution from 1985 to 1997. RESULTS: Of the 198 patients, 15 (7.6%) were found to have a penetrating aortic ulcer on re-review of computed tomographic scans, magnetic resonance images, angiograms, echocardiograms, intraoperative findings, or pathology reports. Two ulcers (13.3%) were located in the ascending aorta; the other 13 (86.7%) were in the descending aorta. In comparison with those with type A or B aortic dissection, patients with penetrating ulcer were older (mean age 76.6 years, p = 0.018); had larger aortic diameters (mean diameter 6.5 cm); had ulcers primarily in the descending aorta (13 of 15 patients, 86.7%); and more often had ulcers associated with a prior diagnosed or managed AAA (6 of 15 patients, 40.0%; p = 0.0001). Risk for aortic rupture was higher among patients with penetrating ulcers (40.0%) than patients with type A (7.0%) or type B (3.6%) aortic dissection (p = 0.0001). CONCLUSIONS: Accurate recognition and differentiation of penetrating ulcers from classic aortic dissection at initial presentation is critical for optimal treatment of these patients. For penetrating ulcer, the prognosis may be more serious than with classic type A or B aortic dissection. Surgical management is advocated for penetrating ulcers in the ascending aorta and for penetrating ulcers in the descending aorta that exhibit early clinical or radiologic signs of deterioration.

Cesarean scar pregnancy: imaging and treatment with conservative surgery.

BACKGROUND: Pregnancy in a cesarean scar represents a rare type of secondary abdominal pregnancy. Early diagnosis can be challenging and optimal treatment is unknown. CASE: A 21-year-old woman presented for an abortion at 8 weeks' gestation. A cesarean delivery had been performed 5 months earlier. Suspecting a cervical pregnancy, her physician referred her to us, and an 8-week cesarean scar gestation was diagnosed and then confirmed by serial sonograms, cystoscopy, and magnetic resonance imaging. The patient elected pregnancy termination, which was accomplished by hysterotomy with uterine preservation followed by intramuscular methotrexate. CONCLUSION: We report a case of cesarean scar pregnancy treated surgically with uterine preservation. This approach should be considered when cesarean scar ectopic pregnancy is diagnosed.

TSH crosslinks to the TSH receptor through the beta subunit.

TSH binds specifically to the TSH receptor present on the surface of thyroid follicular cells and consequently activates adenylate cyclase. It was not known, however, which of the two subunits of the TSH bound to the receptor. By covalently crosslinking TSH to its receptor and characterizing the product with antisera specific for either the alpha or beta subunit of TSH, we have shown that the TSH beta subunit but not the TSH alpha subunit crosslinks to the TSH receptor.

The beta subunits of glycoprotein hormones. Formation of three-dimensional structure during cell-free biosynthesis of lutropin-beta.

The folding of the hormone-specific (beta) subunit of the glycoprotein hormone bovine lutropin was studied after the translation and processing of bovine pituitary mRNA in a system derived from Krebs ascites tumor cells. Of the three forms of beta subunit recognized in this system, only the subunit which had both its prepeptide removed and an oligosaccharide moiety attached formed a tertiary structure which could be immunoprecipitated by an antiserum specific to isolated (folded) lutropin-beta. This glycosylated subunit also combined with added alpha subunit to form the dimeric alpha-beta complex. The results of the translation and processing experiments parallel those of previous experiments in which alpha subunit folding was examined. In contrast to alpha subunit, however, the difficulty of demonstrating correct refolding of beta subunit after reduction and reoxidation of its disulfide bonds strongly suggests that the formation of its correct tertiary structure not only requires carbohydrate attached to the peptide chain but also must occur during formation of the nascent beta chain.

Glycosylation of ovine prolactin during cell-free biosynthesis.

Recently, a glycosylated form of ovine PRL (oPRL) was isolated from a crude pituitary preparation. As glycosylation of PRL was unexpected and because the composition of the oligosaccharide-containing peptide indicated the carbohydrate portion to be extensively degraded, studies of the glycosylation of oPRL during cell-free biosynthesis were initiated. Two glycosylated forms of oPRL can be recognized when biosynthesis occurs in ovine pituitary microsomes. Both forms are converted to mature PRL by digestion with endoglycosidase H and, thus, appear to contain only asparagine-linked, high mannose-type carbohydrate moieties. In contrast, immunoprecipitates from bovine pituitary microsomes consist of the expected (and nonglycosylated) pre-PRL and PRL. The results are consistent with the absence of a sequence segment in the bovine hormone which permits glycosylation (Asn-X-Ser- or Thr-) and the presence of the segment Asn31-Leu-Ser- in the ovine hormone. The occurrence of two glycosylated forms of oPRL is not understood; it may result from an additional site in oPRL capable of glycosylation.

The common alpha subunit of bovine glycoprotein hormones: limited formation of native structure by the totally nonglycosylated polypeptide chain.

The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

Tyrosine residues of bovine thyrotropin. Accessibility to iodination in the intact hormone and isolated subunits.

The susceptibility of the tyrosines of bovine thyrotropin (bTSH) and of its isolated alpha and beta subunits to lactoperoxidase catalyzed iodination was examined as a probe of the reactivity of these residues. In isolated TSH alpha, tyrosines at positions 21, 41, 92 and 93 were labeled with radioactive iodine to a nearly equivalent extent while residue 30 did not incorporate iodine. In intact TSH tyrosine 41, as well as 30, was not modified showing that, upon association with beta subunit, residue 41 becomes substantially less reactive. The other three tyrosines of the alpha subunit retained reactivity; residue 21 incorporated relatively more label than residues 92 and 93. The pattern of reactivity of the TSH alpha tyrosines in intact TSH parallels previous studies on the alpha subunits of lutropin (LH) and human choriogonadotropin (hCG). Thus the different primary sequences of the beta subunits do not influence environments around tyrosines at positions which are spaced throughout the alpha subunit structure. Most of the 11 tyrosine residues of TSH beta incorporate significant amounts of iodine. In intact TSH, beta tyrosines 25, 37, and 119 are most readily iodinated and tyrosines 14, 25 and 37 are the most reactive in the isolated subunit. Of particular interest in intact TSH is that tyrosine 37 and to a lesser extent 61 are modified. These tyrosines are found in analogous and highly conserved domains in the beta subunits of all glycoprotein hormones whose sequences have been determined, but in LH and hCG they can only be iodinated in isolated beta subunits.

Rapid and easy separation of the subunits of bovine and human glycoprotein hormones by use of high performance liquid chromatography.

A single method of reverse phase high performance liquid chromatography is used to separate the subunits of human and bovine glycoprotein hormones. This rapid and easy method is applicable for the separation and detection of subunits from as little as 10 micrograms hormone or the isolation of subunits from as much as 100 mg hormone. Separation is achieved by chromatography on a Vydac 218TP1010 column with a linear (60-min) gradient of 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide to a solvent containing 50% acetonitrile and 50% 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide. Although in some cases the interaction between the hydrophobic support and the hormone is sufficient for dissociation, preincubation of the hormone with guanidine hydrochloride ensures optimum dissociation and improves resolution of the subunits. The subunits isolated by high performance liquid chromatography are functional in that they will reassociate with their counterpart subunits.

Free alpha-like material from bovine pituitaries. Removal of its O-linked oligosaccharide permits combination with lutropin-beta.

Further characterization of the free alpha subunit immunoreactive material, not combined with beta subunit in extracts of bovine pituitaries, shows that the only significant modifications, relative to alpha subunits themselves, are the oligosaccharide O-linked to threonine-43, and heterogeneity of the carboxyl terminus. Removal of the O-linked carbohydrate with a mixture of glycosidases from Streptococcus pneumoniae results in an alpha-like material capable of combining with lutropin beta subunit and, thus, the presence of the oligosaccharide is responsible for the inability of the free alpha-like material to combine with beta subunits. Amino acid compositions of tryptic peptides spanning the entire sequence indicate no change in amino acid sequence of the free alpha-like material as compared to lutropin alpha. Further, based on the similar behavior reverse phase high performance liquid chromatography of the tryptic peptides as compared to their lutropin alpha counterparts, it is concluded that no additional post-translational modifications are present. The N-linked oligosaccharides of the free alpha-like material most likely contain terminal O-sulfated N-acetylhexosamines (as do the asparagine-linked carbohydrates from the pituitary hormones) as indicated by the presence of 3 mol of sulfate/mol of free alpha-like material and the resistance of these oligosaccharides to enzymatic deglycosylation. The O-linked oligosaccharide does not contain sulfated residues.

Binding and degradation of doubly radioiodinated luteinizing hormone by mouse Leydig cells.

Doubly radioiodinated LH (**LH) was used to examine the fate of both subunits during interaction with freshly isolated Leydig cells. Cells that had bound **LH and were then resuspended in **LH-free medium released radioactivity continuously from both subunits in acid-soluble form, but to only a limited extent in acid-precipitable form. With cells from BALB/c mice, the initial rate of release of acid-soluble radioactivity was substantially greater from alpha-subunit than from beta-subunit; this difference was not apparent with cells from Swiss-Webster mice. Appearance of acid-soluble radioactivity was inhibited by leupeptin; testosterone production was not affected. Cell-associated radioactivity declined when the resuspension medium contained unlabeled LH, but assumed a steady state when cells were incubated continuously in **LH. Thus, upon binding of LH to receptor, both subunits are internalized and degraded within the lysosome. Binding and degradation can proceed simultaneously, yet independently. LH degradation has no role in acute testosterone production.

Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance.

Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C-2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (alpha or beta) have had their accessible histidines exchanged with deuterium, permitted assignment of C-2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine alpha-83; in isolated alpha subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit-subunit interaction.

Purification of an alternate form of the alpha subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharide.

Extracts of bovine anterior pituitary glands contain significant amounts of material with immunological properties similar to those of the common, alpha, subunit isolated from the pituitary glycoprotein hormones. Purification of this "free alpha-like" material and analysis show it to contain an additional site of glycosylation not present in the alpha subunit isolated from intact glycoprotein hormones. This additional oligosaccharide is O-linked to a threonine residue corresponding to threonine-43 of bovine lutropin-alpha. Carbohydrate analysis shows 1.7 mol of sialic acid, 0.8 mol of galactose and 0.9 mol of galactosamine/mol of oligosaccharide. A similar structure for the free alpha-like material as compared to bovine lutropin-alpha is evident from equal potency in an anti-lutropin-alpha radioimmunoassay, a similar amino acid composition and similar but not identical peptide maps. The free alpha-like material is distinct from lutropin-alpha in that the free alpha-material contains sialic acid and galactose, has a slightly higher apparent molecular weight, an increased negative charge, and will not reassociate with native lutropin-beta. Peptide maps of the tryptic peptides of the free alpha-like material show additional differences (other than the O-linked oligosaccharide) when compared to peptide maps of lutropin-alpha; thus additional modifications are probably present.

Enzymatic deglycosylation of the subunits of chorionic gonadotropin. Effects on formation of tertiary structure and biological activity.

Both the O- and N-linked oligosaccharide moieties of the subunits of the placental glycoprotein hormone, human choriogonadotropin (hCG), are removed by treatment with a mixture of glycosidases produced by Streptococcus (Diplococcus) pneumoniae. The resulting deglycosylated subunits recombine with their native counterparts in good yield, and the reassociated hormones bind to gonadotropin receptors equally as well as the untreated hormone. Stimulation of steroidogenesis by the deglycosylated alpha-native beta recombinant, however, was markedly less than the stimulation by unmodified hCG both in terms of relative potency (0.10-0.15) and the maximal amount of steroid (40-50%) produced. The native alpha-deglycosylated beta recombinant produced a maximum level of steroid production of 80-90% that of control hCG although its relative potency had decreased approximately 4-fold. The data are in accord with results by others in which either hCG or lutropin was partially deglycosylated by treatment with anhydrous hydrofluoric acid. In addition, the effects of deglycosylation on the ability of each subunit to refold after reduction of their disulfide bonds was studied. Of particular interest is that, after deglycosylation, the beta subunit can correctly refold to a significant degree, in contrast to several unsuccessful attempts to demonstrate correct refolding of the unmodified beta subunit of either lutropin or hCG. Alpha subunit, as measured by a conformation sensitive radioimmunoassay, refolds with equal facility both before and after deglycosylation.

Doubly radioiodinated luteinizing hormone: preparation and characterization.

Bovine [131I]Iodo-alpha LH-[125I]iodo-beta LH (**LH) has been prepared and shown to be physically and biologically equivalent to unmodified hormone. The beta-subunit was modified with 125I, purified by adsorption to Concanavalin A-Sepharose and elution with methylmannoside, added to alpha-subunit, and allowed to reassociate to intact hormone. Iodination with 131I was then carried out in the reassociation mixture and **LH was isolated by gel filtration. Both gel electrophoresis and rechromatography on Sephadex G-100 showed that both radiolabels comigrated with unmodified hormone. Sodium dodecyl sulfate gel electrophoresis showed that 131I was found in the alpha-subunit and 125I in the beta-subunit; this result is in agreement with studies by others which show that the tyrosines of the beta-subunit are nonreactive in intact hormone. In receptor-binding assays, both radiolabels were specifically displaced in a similar fashion by LH. Scatchard analysis showed high affinity binding (Ka approximately equal to 1.5 X 10(10) M-1) for both labels. Comparison of receptor-binding activity with steroidogenic activity showed that iodinated hormone molecules not only bound to receptor but also stimulated testosterone production. The demonstration that full biological activity is retained with iodination in both subunits shows that such doubly labeled LH can be used to monitor the disposition of both subunits simultaneously during interaction of the hormone with target cells.