Effects of conventional and alternative curing methods on processed turkey quality traits.

Deli-style turkey breast cured with pre-converted celery juice powder (CP) or sodium nitrite (SN) was manufactured with ingoing SN concentrations or equivalent of 0, 50, 100, 150, and 200 ppm. Cured and total meat pigment, salt, and water activity were measured on d 0; color, pH, and residual nitrite were measured on d 0, 7, 14, 21, 28, 35, and 42. Untrained panelists evaluated sensory traits of 50, 100, 150, and 200 ppm products. The interaction of nitrite concentration and source affected (P≤0.05) b*, pH, and residual nitrite values. Less ingoing nitrite and increased storage resulted in less (P≤0.05) residual nitrite in products. Sensory results suggested products made with 200 ppm nitrite from CP were less acceptable. Overall, CP and SN products were similar for several traits, but this study suggests that the inclusion of ingoing nitrite from CP may be limited to 100 ppm nitrite (0.46% addition) for acceptable deli-style turkey breast.

Analog filtering of large solvent signals for improved dynamic range in high-resolution NMR.

The large solvent signal from samples in H2O solvent still challenges the dynamic range capability of any spectrometer. The solvent signal can be largely removed with a pair of simple resistor-capacitor (RC) high-pass filters when the solvent frequency is set at center band (zero frequency) using quadrature detection, with RC approximately 0.5 ms. However, an approximately 0.5-ms transient remains at initial time, which we reduce fourfold for a short time only, just before the A/D converter, by means of a variable-gain amplifier, and later restore with software. This modification can result in a nearly fourfold increase in dynamic range. When we converted to a frequency-shifted mode (A. G. Redfield and S. D. Kunz, 1994, J. Magn. Reson. A 108, 234-237) we replaced the RC high-pass filter with a quadrature feedback notch filter tuned to the solvent frequency (5.06 kHz). This filter is an example of a class of two-input/two-output filters which maintain the spectral integrity (image-free character) of quadrature signals. Digital filters of the same type are also considered briefly. We discuss the implications of these ideas for spectrometer input design, including schemes for elimination of radiation damping, and effects of probe bandwidth on extreme oversampling.

Cadherins promote skeletal muscle differentiation in three-dimensional cultures.

The cell-cell adhesion molecule N-cadherin, with its associated catenins, is expressed by differentiating skeletal muscle and its precursors. Although N-cadherin's role in later events of skeletal myogenesis such as adhesion during myoblast fusion is well established, less is known about its role in earlier events such as commitment and differentiation. Using an in vitro model system, we have determined that N-cadherin- mediated adhesion enhances skeletal muscle differentiation in three-dimensional cell aggregates. We transfected the cadherin-negative BHK fibroblastlike cell line with N-cadherin. Expression of exogenous N-cadherin upregulated endogenous beta-catenin and induced strong cell-cell adhesion. When BHK cells were cultured as three-dimensional aggregates, N-cadherin enhanced withdrawal from the cell cycle and stimulated differentiation into skeletal muscle as measured by increased expression of sarcomeric myosin and the 12/101 antigen. In contrast, N-cadherin did not stimulate differentiation of BHK cells in monolayer cultures. The effect of N-cadherin was not unique since E-cadherin also increased the level of sarcomeric myosin in BHK aggregates. However, a nonfunctional mutant N-cadherin that increased the level of beta-catenin failed to promote skeletal muscle differentiation suggesting an adhesion-competent cadherin is required. Our results suggest that cadherin-mediated cell-cell interactions during embryogenesis can dramatically influence skeletal myogenesis.

Conformational and dynamic differences between N-ras P21 bound to GTPgammaS and to GMPPNP as studied by NMR.

Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational changes between the GMPPNP form of human N-ras P21 as compared to GDP and GTPgammaS forms. Full-length N-ras P21 was also compared with protein truncated beyond residue 167, to search for interaction points between the more invariant part of the protein and the variable C-terminal section. In both cases, the reporter was the 15N-H 2D spectrum of aspartate amide groups labeled with 15N. Small truncation-induced changes were seen in the spectrum at the resonances of Asp-54, -108, and -109 which are not far from the C-terminal and, surprisingly, at Asp-57 which is more remote. The spectrum obtained for the GMPPNP-ligated form is similar to that of the GTPgammaS form, except that peaks of several residues are weak at low temperature, and strongly temperature-dependent in their intensity, and a new resonance appears at 15 degrees C and above. The observations are discussed in terms of a two-state model for the GMPPNP-ligated protein, previously proposed by Geyer et al. [(1996) Biochemistry 35, 10308-10320].

Characterization of the interactions of alpha-catenin with alpha-actinin and beta-catenin/plakoglobin.

Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin and gamma-catenin (also known as plakoglobin), have been identified. beta-catenin or plakoglobin is associated directly with the cadherin; alpha-catenin binds to beta-catenin/plakoglobin and serves to link the cadherin/catenin complex to the actin cytoskeleton. The domains on the cadherin and betacatenin/plakoglobin that are responsible for protein-protein interactions have been mapped. However, little is known about the molecular interactions between alpha-catenin and beta-catenin/plakoglobin or about the interactions between alpha-catenin and the cytoskeleton. In this study we have used the yeast two-hybrid system to map the domains on alpha-catenin that allow it to associate with beta-catenin/plakoglobin and with alpha-actinin. We also identify a region on alpha-actinin that is responsible for its interaction with alpha-catenin. The yeast two-hybrid data were confirmed with biochemical studies.

General stress transcription factor sigmaB of Bacillus subtilis is a stable protein.

The sigmaB subunit of Bacillus subtilis RNA polymerase governs the expression of a large general stress regulon. The results of pulse-chase and immunoprecipitation experiments showed that sigmaB is stable both in the presence and in the absence of the RsbW anti-sigma factor, the principal regulator of sigmaB in response to environmental signals.

The effect of 17O on the relaxation of an amide proton within a hydrogen bond.

The relaxation rates of the multiple-quantum coherence for the amide hydrogen of Gly13 in ras p21.GDP were determined in the presence and absence of 17O labeling in the beta-phosphate of GDP. No significant difference could be observed between labeled and unlabeled samples, in spite of the fact that the hydrogen bond from the amide group of Gly13 to the beta-phosphate is shorter than is typical, based on its chemical shift. For macromolecules in which an oxygen atom is the acceptor of a hydrogen bond, dipolar coupling between 17O and hydrogen is unlikely to be observable, except for extremely short H-bonds.

Proton exchange and basepair kinetics of yeast tRNA(Phe) and tRNA(Asp1).

Nuclear magnetic resonance measurements of proton exchange were performed on yeast tRNA(Phe) and yeast tRNA(Asp), at temperatures from 20 to 45 degrees C, in the presence of various levels of salt, phosphate, added magnesium, and pH. The dynamical changes of the tRNA molecule were interpreted, with the aid of firmly established assignments and the use of the saturation recovery technique. In tRNA(Phe), the exchange rates in zero magnesium indicated early melting of the acceptor stem, tertiary structure, and D stem. However, in the presence of even low levels of magnesium the D stem remained intact up to high temperature, stabilized by a Mg2+ ion. A similar unfolding sequence was observed in tRNA(Asp). The difference between these two tRNAs was the thermal behavior of the tertiary resonance U8-A14. In tRNA(Phe), this base pair showed sharp rate increases between 32 and 39 degrees C. However, in tRNA(Asp), it remained intact up to 36 degrees C and disappeared at 39 degrees C, even if there was not important kinetic broadening. By measuring the temperature dependence of the exchange rates, we obtained an activation energy of 40-60 kcal/mol for all the imino protons of yeast tRNA(Phe) in zero magnesium. The same activation energy was obtained for tRNA(Phe) with equimolar concentration of magnesium. By investigation of the dependence of the exchange rates of these imino protons on solution conditions, we observed the transition from kop rate limiting in the absence of magnesium to kex rate limiting in the presence of magnesium.

Effects of the angiotensin II antagonist losartan in hypertensive patients with renal disease.

OBJECTIVE: To study the effects of the angiotensin II antagonist losartan in hypertensive patients with renal disease. DESIGN: A single-blind longitudinal study was performed, lasting 4 months. Patients were treated once a day with placebo, 50 mg losartan, 100 mg losartan and placebo, each for 1 month, in sequence. Measurements were taken at the end of each treatment period. PATIENTS: The study subjects comprised 13 patients with biopsy-proven renal disease, a diastolic blood pressure of > 90 mmHg, creatinine clearance of > 60 ml/min and stable proteinuria of > 2.0 g/day. RESULTS: Mean arterial pressure, measured 4 h after the dose, fell from 113.8 +/- 2.1 mmHg (placebo) to 99.2 +/- 2.4 mmHg (50-mg dose) and 96.5 +/- 2.4 mmHg (100-mg dose). This blood pressure lowering effect was sustained for 24 h 116.3 +/- 2.3 mmHg (placebo) versus 105.5 +/- 1.8 mmHg (50-mg dose) and 103.3 +/- 1.9 mmHg (100-mg dose)]. The glomerular filtration rate remained stable, while the effective renal plasma flow increased by 12.5 +/- 2.9% (100-mg dose). The systemic and renal hemodynamic effects were similar at the 50- and 100-mg doses. Urinary excretion of total protein, albumin and immunoglobulin G decreased dose-dependently by a maximum of +/- 50% (100-mg dose). With the high dose, serum uric acid fell from 0.43 +/- 0.02 mmol/l to 0.39 +/- 0.02 mmol/l, and potassium increased from 4.2 +/- 0.1 to 4.6 +/- 0.1 mmol/l. CONCLUSIONS: The angiotensin II antagonist losartan lowers blood pressure and displays a favorable renal profile in hypertensive patients with renal disease.

Characterization of the active site of p21 ras by electron spin-echo envelope modulation spectroscopy with selective labeling: comparisons between GDP and GTP forms.

Selectively labeled samples of human H- or N-ras p21 ligated to MnIIGDP or MnIIGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated [4-13C]-labeled Asx into p21.MnIIGDP and found that the distance from the carboxyl 13C of Asp57 to MnII is approximately 4.1 A. Our result is consistent with indirect coordination of this residue to the metal. From a [2-2H]Thr-labeled sample, we estimate that the distance from the MnII ion to the 2H of Thr35 is at least 5.8 A. Thus, the only protein or nucleotide ligands to the metal appear to be Ser17 and the beta-phosphate of GDP, as previously reported [Larsen, R. G., Halkides, C. J., Redfield, A. G., & Singel, D. J. (1992b) J. Am. Chem. Soc. 114, 9608-9611]. In the 5'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been reported by X-ray crystallography to be a ligand of the metal via its hydroxyl group, and this residue appears to play a key role in the biologically important conformational change upon nucleotide substitution [Pai, E. F., Krengel, U., Petsko, G., Goody, R. S., Kabsch, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The ESEEM spectrum of p21.MnIIGMPPNP labeled with [2-2H]Thr yields a MnII-2H distance of 4.9 A, a distance inconsistent with strong coordination. A sample of p21 in which the Thr residues were fully labeled with 13C and 15N yielded a value of 5.0 A for the distance from MnII to the amide nitrogen of Thr35, while the 13C signal is much smaller than expected if Thr35 were coordinated. A [15N]serine/glycine-labeled sample gives a distance to the amide 15N of Ser17 of 3.9 A, consistent with the X-ray structure; a [4-13C]-labeled Asx sample of p21 gives a distance of approximately 4 A between MnII and the label of Asp57, again implying indirect coordination. Both of these values are very similar to those found for the GDP form of the protein. The results for Thr35, however, reveal a structural difference between the GDP and GTP forms in the region of Thr35. In addition, the position of this residue is found to be different from the crystal structure and in a manner suggesting that the metal ligation of Thr35 does not drive the conformational change that accompanies nucleotide substitution.

Stress-induced activation of the sigma B transcription factor of Bacillus subtilis.

The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals. On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions. Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response. After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression. Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth. Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells. However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus.

An NMR comparison of the changes produced by different guanosine 5'-triphosphate analogs in wild-type and oncogenic mutant p21ras.

We have used nuclear magnetic resonance spectroscopy to compare the conformational changes produced by replacement of bound GDP by the GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanylyl (beta, gamma-imido)diphosphate (GMPPNP) in wild-type p21ras as well as the oncogenic mutant (G12D)p21ras. We have used isotope-edited nuclear magnetic resonance spectroscopy to observe the amide resonances of selectively [15N]glycine and [15N]isoleucine labeled p21ras-nucleotide complexes. We find that eight of the nine resonances that respond strongly to GTP gamma S and GMPPNP binding are the same but that the nature of the effect appears different. With GTP gamma S, seven new resonances replace the eight resonances specifically associated with GDP-p21ras, but in GMPPNP-p21ras only two resonances replace the GDP-specific resonances that are lost. The resonance of Gly 60 is clearly shown to be responsive to replacement of GDP by GMPPNP, in addition to glycines 10, 12, 13, 15, and 75 and isoleucines 36, 21, and one other, that were found to respond to GTP gamma S by Miller et al. [Miller, A.-F., Papastavros, M. Z., & Redfield, A.G. (1992) Biochemistry 31, 10208-10216). The two GMPPNP-specific resonances observed appear in positions similar to GTP gamma S-specific resonances, and the GTP gamma S-specific resonances, although not lost altogether, are weaker than the GDP-specific resonances they replace. Thus, the two GTP analogs have similar effects on the spectrum of p21ras, suggesting that the effects are due to features common to both analogs. We propose that active site resonance intensities are specifically attenuated when GTP analogs are bound because interactions with the gamma-phosphate of GTP analogs couple the flexible loops 2 and 4 to the rigid loop 1 of the active site. The conformational heterogeneity and dynamics of loops 2 and 4 would be constrained by loop 1 but also transmitted to it. Coupled conformational exchange on a common intermediate time scale could explain the simultaneous loss of resonances from all three loops in the active site. In our comparison of wild-type and (G12D) GDP-p21ras, we find that the resonance of Ile 36 is not visible in (G12D)p21ras. In (G12D)p21ras, replacement of GDP by GTP gamma S causes the resonances of glycines 10, 13, 15, 60, and 75 and isoleucine 21 and four others to shift from their GDP-specific positions. GTP gamma S-specific resonances are observed for all but two of these.(ABSTRACT TRUNCATED AT 400 WORDS)

Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon.

Transcription factor sigma B of Bacillus subtilis is active during the stationary growth phase, but its physiological role remains unknown. Understanding the function and regulation of genes controlled by sigma B (csb genes) should provide important clues to sigma B function in stationary-phase cells. To this end, we used a genetic approach to identify six new csb genes. This strategy relies on two elements: (i) random transcriptional fusions between the Escherichia coli lacZ gene and genes on the B. subtilis chromosome, generated in vivo with transposon Tn917lacZ, and (ii) a plate transformation technique to introduce a null sigB mutation into the fusion-bearing recipients directly on indicator plates. This strategy allowed the comparison of fusion expression in strains that were isogenic save for the presence or absence of a functional sigma B protein. Beginning with 1,400 active fusions, we identified 11 that were wholly or partly controlled by sigma B. These fusions mapped to six different loci that exhibit substantial contrasts in their patterns of expression in the logarithmic and stationary growth phases, suggesting that they participate in diverse cellular functions. However, for all six loci, the sigma B-dependent component of their expression was manifest largely in the stationary phase. The high frequency of six independent csb loci detected in a random collection of 1,400 fusions screened, the fact that four of the six new loci were defined by a single fusion, and the absence of the previously identified ctc and csbA genes in the present collection strongly suggest that sigma B controls a large stationary-phase regulon.

Mapping the nucleotide-dependent conformational change of human N-ras p21 in solution by heteronuclear-edited proton-observed NMR methods.

Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational change between GDP- and GTP gamma S-bound forms of human N-ras p21. Amide groups of Asp are used as sensitive probes. When GTP gamma S is substituted for GDP in cellular N-ras p21, the chemical shifts of resonances Asp-47, -126, -154, and Asn-172, as well as Gly-77 and -151, are not sensitive to nucleotide exchange, whereas Asp-30, -33, -38, -54, -57, -69, -92, -105, and -119 are affected. Distinct chemical shift changes of Asp-33, -38, and -69 indicate that substantial structure changes occur in the effector-binding region and the switch II region. Crystallographic studies of H-ras p21 have indicated that the conformational differences are confined to residues 32-38 and 60-76. Our observations indicate that the nucleotide-dependent structural transitions of the protein in solution may not be identical to those in the crystal. They suggest that the peptide beyond Glu-76 participates in a conformational switch, and possibly is involved in effector function. We propose that the region roughly from Asp-92 to -105, and the region of guanine base-binding motif(s), e.g., 116NKXD, are candidate sites recognized by either a GDP/GTP release factor or a GTPase-affected protein.

NMR study of nitrogen-15-labeled Escherichia coli valine transfer RNA.

1,3-15N-Labeled uracil was synthesized chemically and used to prepare labeled Escherichia coli tRNA(Val) biosynthetically. 500-MHz measurements of 15N and proton chemical shift were obtained, for all uridine and uridine-related bases, by heteronuclear multiple-quantum coherence spectroscopy. All the uracil NH group resonances were assigned and were in agreement with previous proton-only assignments. The temperature dependence of intensities of resonances was used to infer the relative stability of parts of the molecule. The acceptor stem was the least thermally stable structural feature, while the anticodon and T loop were relatively more stable.

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.

NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain.

The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods. Five proton resonances were found below 10.5 ppm. Two of these were assigned to the amide groups of Lys 24 and Gly 83. These are conserved residues in each of the consensus sequences. Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 [Redfield, A. G., & Papastavros, M. Z. (1990) Biochemistry 29, 3509-3514]. The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21. These results suggest that this conserved lysine has a similar structural role in proteins in this class. The tentative Gly 83 resonance has no spectral analogue in p21. A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.

Molecular structure of charybdotoxin: retraction.

Shortly after our paper of 3 August 1990 on the molecular structure of charybdotoxin (1) was published, two independent determinations of the structure of this molecule appeared (2) that were similar to each other and in strong disagreement with ours. We have obtained new data and find that some spectral features depend on solvent conditions, which explains some differences between our data and those of the other groups. More important, we conclude that we most probably misassigned an important sequence of amino acids, as suggested by Bontems et al.(3). Therefore, we withdraw our previously reported structure (1) and regret any inconvenience it may have caused. We thank F. Toma for sending us a copy of his paper before publication and for discussions.

Molecular structure of charybdotoxin, a pore-directed inhibitor of potassium ion channels.

The three-dimensional structure of charybdotoxin, a high-affinity peptide blocker of several potassium ion channels, was determined by two-dimensional nuclear magnetic resonance (2-D NMR) spectroscopy. Unambiguous NMR assignments of backbone and side chain hydrogens were made for all 37 amino acids. The structure was determined by distance geometry and refined by nuclear Overhauser and exchange spectroscopy back calculation. The peptide is built on a foundation of three antiparallel beta strands to which other parts of the sequence are attached by three disulfide bridges. The overall shape is roughly ellipsoidal, with axes of approximately 2.5 and 1.5 nanometers. Nine of the ten charged groups are located on one side of the ellipsoid, with seven of the eight positive residues lying in a stripe 2.5 nanometers in length. The other side displays three hydrophobic residues projecting prominently into aqueous solution. The structure rationalizes several mechanistic features of charybdotoxin block of the high-conductance Ca2(+)-activated K+ channel.