Spatial and temporal inhibition of FGFR2b ligands reveals continuous requirements and novel targets in mouse inner ear morphogenesis.

Morphogenesis of the inner ear epithelium requires coordinated deployment of several signaling pathways, and disruptions cause abnormalities of hearing and/or balance. The FGFR2b ligands FGF3 and FGF10 are expressed throughout otic development and are required individually for normal morphogenesis, but their prior and redundant roles in otic placode induction complicates investigation of subsequent combinatorial functions in morphogenesis. To interrogate these roles and identify new effectors of FGF3 and FGF10 signaling at the earliest stages of otic morphogenesis, we used conditional gene ablation after otic placode induction, and temporal inhibition of signaling with a secreted, dominant-negative FGFR2b ectodomain. We show that both ligands are required continuously after otocyst formation for maintenance of otic neuroblasts and for patterning and proliferation of the epithelium, leading to normal morphogenesis of both the cochlear and vestibular domains. Furthermore, the first genome-wide identification of proximal targets of FGFR2b signaling in the early otocyst reveals novel candidate genes for inner ear development and function.

Fgf3 is required for dorsal patterning and morphogenesis of the inner ear epithelium.

The inner ear, which contains sensory organs specialized for hearing and balance, develops from an ectodermal placode that invaginates lateral to hindbrain rhombomeres (r) 5-6 to form the otic vesicle. Under the influence of signals from intra- and extraotic sources, the vesicle is molecularly patterned and undergoes morphogenesis and cell-type differentiation to acquire its distinct functional compartments. We show in mouse that Fgf3, which is expressed in the hindbrain from otic induction through endolymphatic duct outgrowth, and in the prospective neurosensory domain of the otic epithelium as morphogenesis initiates, is required for both auditory and vestibular function. We provide new morphologic data on otic dysmorphogenesis in Fgf3 mutants, which show a range of malformations similar to those of Mafb (Kreisler), Hoxa1 and Gbx2 mutants, the most common phenotype being failure of endolymphatic duct and common crus formation, accompanied by epithelial dilatation and reduced cochlear coiling. The malformations have close parallels with those seen in hearing-impaired patients. The morphologic data, together with an analysis of changes in the molecular patterning of Fgf3 mutant otic vesicles, and comparisons with other mutations affecting otic morphogenesis, allow placement of Fgf3 between hindbrain-expressed Hoxa1 and Mafb, and otic vesicle-expressed Gbx2, in the genetic cascade initiated by WNT signaling that leads to dorsal otic patterning and endolymphatic duct formation. Finally, we show that Fgf3 prevents ventral expansion of r5-6 neurectodermal Wnt3a, serving to focus inductive WNT signals on the dorsal otic vesicle and highlighting a new example of cross-talk between the two signaling systems.

Effects of dietary carbohydrate on delayed onset muscle soreness and reactive oxygen species after contraction induced muscle damage.

BACKGROUND: Delayed onset muscle soreness (DOMS) occurs after unaccustomed exercise and has been suggested to be attributable to reactive oxygen species (ROS). Previous studies have shown increased ROS after lengthening contractions, attributable to invading phagocytes. Plasma glucose is a vital fuel for phagocytes, therefore carbohydrate (CHO) status before exercise may influence ROS production and DOMS. OBJECTIVE: To examine the effect of pre-exercise CHO status on DOMS, ROS production, and muscle function after contraction induced muscle damage. METHOD: Twelve subjects performed two downhill runs, one after a high CHO diet and one after a low CHO diet. Blood samples were drawn for analysis of malondialdehyde, total glutathione, creatine kinase, non-esterified fatty acids, lactate, glucose, and leucocytes. DOMS and muscle function were assessed daily. RESULTS: The high CHO diet resulted in higher respiratory exchange ratio and lactate concentrations than the low CHO diet before exercise. The low CHO diet resulted in higher non-esterified fatty acid concentrations before exercise. DOMS developed after exercise and remained for up to 96 hours, after both diets. A biphasic response in creatine kinase occurred after both diets at 24 and 96 hours after exercise. Malondialdehyde had increased 72 hours after exercise after both diets, and muscle function was attenuated up to this time. CONCLUSIONS: Downhill running resulted in increased ROS production and ratings of DOMS and secondary increases in muscle damage. CHO status before exercise had no effect.

Evaluating substance abuse treatment process models: I. Changes on proximal outcome variables during 12-step and cognitive-behavioral treatment.

OBJECTIVE: This article provides data on the early linkages in the treatment process chains that are thought to underlie two prevalent approaches to substance abuse treatment-traditional 12-step treatment and cognitive-behavioral treatment. The focus is on the during-treatment changes on "proximal outcomes" that, according to the treatment theory underlying each modality, patients are supposed to undergo or achieve in order to experience a positive "ultimate outcome." METHOD: In all, 3,228 men receiving treatment in 15 Department of Veterans Affairs substance abuse treatment programs were assessed at treatment entry and at or near discharge from inpatient programs that had desired lengths of stay of 21-28 days. RESULTS: Between intake and discharge, patients in 12-step programs improved more than did C-B patients on proximal outcome variables assumed to be specific to 12-step treatment (e.g., attending 12-step meetings, taking steps), whereas patients in cognitive-behavioral programs made no greater change (and in a few cases, less change) than did 12-step patients on proximal outcome variables assumed to underlie cognitive-behavioral treatment (e.g., self-efficacy, coping skills). CONCLUSIONS: These findings suggest that the proximal outcomes thought to be specific to cognitive-behavioral treatment are actually general proximal outcomes of both 12-step and cognitive-behavioral treatment.

Structural and functional properties of human alpha-thrombin, phosphopyridoxylated alpha-thrombin, and gamma T-thrombin. Identification of lysyl residues in alpha-thrombin that are critical for heparin and fibrin(ogen) interactions.

alpha-Thrombin derivatives obtained either by site-specific modification at lysyl residues (phosphopyridoxylated) or by limited trypsinolysis (gamma T-thrombin) were compared to correlate structural modifications with the functional reactivity toward fibrin(ogen) and heparin. alpha-Thrombin phosphopyridoxylated in the absence of heparin (unprotected) showed approximately 2 mol of label incorporated/mol of thrombin, but only 1 mol of label incorporated/mol of proteinase when modified in the presence of added heparin (protected). In contrast to native alpha-thrombin, both phosphopyridoxylated alpha-thrombin derivatives failed to interact with a fibrin monomer-agarose column and had reduced fibrinogen clotting activity, which is very similar to gamma T-thrombin. Heparin accelerated the rate of antithrombin III inhibition of alpha-thrombin, heparin-protected modified-alpha-thrombin, and gamma T-thrombin in a manner consistent with a template mechanism but was without effect on unprotected modified alpha-thrombin. In a heparin-catalyzed antithrombin III inhibition assay of alpha-thrombin, we found that D-Phe-Pro-Arg chloromethyl ketone-active site-inactivated gamma T-thrombin competed for heparin binding. It has been shown that limited proteolysis/autolysis of the B-chain of alpha-thrombin in the area around Arg-B73 (in beta T/beta- and gamma T/gamma-thrombin), but not that around Lys-B154 (in gamma T/gamma-thrombin), diminishes specific interactions with fibrinogen (Hofsteenge, J., Braun, P. J., and Stone , S. R. (1988) Biochemistry 27, 2144-2151). In unprotected modified alpha-thrombin, lysyl residues B21, B65, B174, and B252 were phosphopyridoxylated. In heparin-protected modified alpha-thrombin, only lysyl residues B21 and B65 were phosphopyridoxylated. These observations suggest that lysyl residues 21/65 of the B-chain of alpha-thrombin are involved in fibrin(ogen) interactions, and lysyl residues 174/252 of the B-chain are important in heparin interactions.

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment.

Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.

The reaction of bovine alpha-thrombin with tetranitromethane. Characterization of the modified protein.

Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is characterized by an apparently greater loss of fibrinogen-clotting activity than activity toward synthetic ester substrates, suggesting that the residues modified by tetranitromethane are involved in the interaction of thrombin with fibrinogen. This study was designed 1) to determine the effect of solvent conditions on the rate of modification and the stoichiometry of the reaction of tetranitromethane with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3) to characterize the modified enzyme with respect to its interaction with peptide nitroanilide substrates and fibrinogen. The inactivation of thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0, than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10% fibrinogen-clotting activity remained at maximal inactivation. A study of the effect of tetranitromethane concentration on the rate of inactivation suggested that the loss of activity was the result of the modification of 1 mol of tyrosine/mol of thrombin. A similar result was obtained from the analysis of the extent of inactivation as a function of the extent of protein modification. Structural analysis of the modified protein showed substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies were performed with the modified protein and a control thrombin with N2-tosylglycylprolylarginine p-nitroanilide. H-D-phenylalanylpipecolylarginine p-nitronailide, and purified bovine fibrinogen. With all three substrates, a substantial decrease in kcat was observed, whereas there was essentially no change in Km. These results suggest that, contrary to previous suggestions, the modification of Tyr71 and Tyr85 in thrombin does not influence the binding of substrates, but rather influences active site reactivity.

Locus of a histidine-based, stable trifunctional, helix to helix collagen cross-link: stereospecific collagen structure of type I skin fibrils.

The loci of the three amino acid residues that contribute their prosthetic groups to form the stable, nonreducible, trifunctional intermolecular cross-link histidinohydroxylysinonorleucine in skin collagen fibrils were identified. Two apparently homogeneous three-chained histidinohydroxylysinonorleucine cross-linked peptides were chromatographically isolated. They were obtained from a tryptic digest of denatured unreduced 6 M guanidine hydrochloride insoluble bovine skin collagen. Amino acid and sequence analyses demonstrated that the prosthetic groups of alpha 1(I)-chain Hyl-87, alpha 1(I)-chain Lys-16c, and alpha 2(I)-chain His-92 formed the cross-link. The latter results served to define the locus of the stable, nonreducible trifunctional moiety. Identical types of analyses were performed on the three-chained peptides isolated after bacterial collagenase digestion of the cross-linked tryptic peptides. This confirmed the initial identification and location of the three peptides linked by the cross-link. In addition, data reported here provide for a correction of the micromolecular structure for the alpha 2(I) chain. Stereochemical considerations concerning this trifunctional cross-link's specific locus indicate that the steric relationships between the alpha chains of skin and skeletal tissue collagens are fundamentally different and the intermolecular relationships in skin fibrils are specific for skin. The same molecular relationships also indicate that histidinohydroxylysinonorleucine links three molecules of collagen. The stereochemistry of cross-linking for skin collagen is in accordance with and explains the X-ray findings of a 65-nm periodicity found for this tissue [Stinson, R. H., & Sweeny, P. R. (1980) Biochim. Biophys. Acta 621, 158; Brodsky, B., Eikenberry, E. F., & Cassidy, K. (1980) Biochim. Biophys. Acta 621, 162].

Identification of a lysyl residue in antithrombin which is essential for heparin binding.

Identification of lysyl residue(s) in human plasma antithrombin required for binding of heparin was approached using chemical modification with the amino-group reagent pyridoxal 5'-phosphate. Modification of antithrombin with limiting amounts of reagent yields an average incorporation of the phosphopyridoxyl label into 1 lysine/protein molecule (Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938). Fractionation of the labeled antithrombin by affinity chromatography on heparin-Sepharose separated a phosphopyridoxylated antithrombin species devoid of heparin binding from modified protein which retained affinity for heparin. To generate peptide maps of the two antithrombin species, the proteins were reductively denatured, S-carboxymethylated, and digested with trypsin. Fractionation of the tryptic digests by reverse-phase high performance liquid chromatography indicated one peak in the chromatogram of the non-heparin-binding species to be clearly different when compared to the chromatogram of the heparin-binding species. The sequence of the unique peptide, determined by automated Edman degradation, was Thr-Ser-Asp-Gln-Ile-His-Phe-Phe-Phe-Ala-Lys-Leu-Asn-Cys-Arg. This peptide corresponds to a tryptic fragment including residues 115-129 in the sequence of antithrombin, with the modified residue identified as Lys-125. Additionally, phosphopyridoxylation of antithrombin in the presence of added heparin indicated that several other lysyl residues were "protected" from modification. Identification of this critical lysine for heparin binding strongly supports previous data which indicate that the heparin-binding domain of antithrombin is located at the NH2 terminus within one of the disulfide cross-linked loops of the protein.

Comparison of five techniques for the determination of protein content in mixed human saliva.

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.

Mechanism of the calcium-dependent self-association of bovine prothrombin. Use of a covalent cross-linking reagent to study the reaction.

The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin and has demonstrated that the covalent dimerization reaction involves the gamma-carboxyglutamic acid region of prothrombin (1-42 of 582). An essential role for the gamma-carboxyglutamic acid residues of prothrombin in the association reaction was demonstrated by experiments that converted gamma-carboxyglutamic acid residues to gamma-methylene glutamic acid or glutamic acid and resulted in a prothrombin species that was inactive in our cross-linking assay. Other experiments showed that very high concentrations of calcium ion inhibit the cross-linkage of prothrombin. This result is most consistent with an essential gamma-carboxyglutamic acid-calcium ion-gamma-carboxyglutamic acid bridge(s) in the calcium-dependent self-associated form of prothrombin.

Structural evidence for leucine at the reactive site of heparin cofactor II.

The reaction products formed during the enzymatic inactivation of heparin cofactor II (HCII) by a proteinase isolated from Echis carinatus were analyzed by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and by reverse-phase high-performance liquid chromatography. By NaDodSO4-polyacrylamide gel electrophoresis, limited proteolysis of HCII was observed, which resulted in a decrease in the apparent molecular weight of the protein from approximately 68 000 to approximately 53 000. By reverse-phase high-performance liquid chromatography, at least 20 peptides were observed. Primary structure analysis of these peptides indicated that significant proteolysis had occurred in the NH2-terminal region of the protein. HCII inactivation, however, coincided with the appearance of a peptide from the COOH-terminal region of the protein. The peptide differed from the previously identified reactive site peptide [Griffith, M. J., Noyes, C. M., & Church, F. C. (1985) J. Biol. Chem. 260, 2218-2225] by only one residue: a leucyl residue at the NH2-terminal of the peptide. We conclude that leucine, as opposed to the expected arginine, is at the reactive site of HCII.

Inhibition of chymotrypsin by heparin cofactor II.

Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermatan sulfate on the rate of chymotrypsin inhibition. Arginine-modified heparin cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofactor II and chymotrypsin formed a stable complex with a Mr value near 90,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis; this suggests a 1:1 reaction stoichiometry. The chymotrypsin cleavage site in heparin cofactor II was the same as that for thrombin, and primary structure analysis of the inhibitor showed a P'1-P'8 sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . The results indicate that, in contrast to alpha 1-antichymotrypsin, which does not inhibit trypsin-like enzymes, including thrombin, heparin cofactor II can effectively inhibit both thrombin and chymotrypsin.

Enzymatic inactivation of heparin cofactor II by a proteinase (proteinase-1) isolated from Echis carinatus venom.

Heparin cofactor II was enzymatically inactivated by incubation with Echis carinatus venom in the presence of calcium. The initial rate of inactivation increased proportionately with the addition of heparin to a final concentration of 50 micrograms/ml. A proteinase, termed proteinase-1, was purified 17.5-fold from the venom which also enzymatically inactivated heparin cofactor II in the presence of calcium. The initial rate of heparin cofactor II inactivation by proteinase-1 was not increased by heparin at concentrations as high as 200 micrograms/ml. Heparin cofactor II was not inactivated by either unfractionated venom or proteinase-1 in the absence of calcium. The results indicate that heparin cofactor II, like antithrombin III, is susceptible to enzymatic inactivation by metalloproteinases in snake venoms.

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin.

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombin's catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.