The effect of feeding ractopamine (Paylean) on muscle quality and sensory characteristics in three diverse genetic lines of swine.

The present experiment utilized Berkshire (n = 76), Duroc (n = 81), and high-lean commercial crossbred (n = 75) barrows and gilts with an initial BW of approximately 85.1 kg. Pigs were fed a standard commercial diet (17.6% CP, 1.02% lysine) supplemented with ractopamine hydrochloride at a level of 0 or 10 ppm for 28 d. The experiment was conducted in a randomized complete block design, with animals blocked within genetic line according to litter, gender, and weight, for a total of four blocks per genetic line for each treatment. Pigs were harvested at a commercial abattoir and chilled for 24 h at 1 to 4 degrees C. At 24 h postmortem, wetness and firmness scores and ultimate muscle pH were measured in the center of the longissimus muscle (LM) at the 10th to 11th rib interface. Visual and instrumental color and marbling score were measured at 48 h postmortem on a fresh cut LM surface. Percentage of chemically extracted intramuscular fat (IMF) was measured, and a trained sensory panel evaluated cooked LM chops for juiciness, tenderness, and chewiness. Cooking loss (%) and instrumental measurement of tenderness also were measured on cooked LM chops. Ractopamine treatment increased ADG (P < 0.01) and LM area (P < 0.05), but had no effect (P > 0.05) on LM quality, sensory attributes, or instrumental measures of palatability. Berkshire LM received higher tenderness and juiciness (P < 0.05) scores and had lower cooking losses (P < 0.05) and instrumental tenderness (P < 0.05) than LM from the Duroc and high-lean lines. Loins from barrows were firmer (P < 0.05), had lower drip loss percentages (P < 0.05), and received greater tenderness scores (P < 0.05) than the LM from gilts. Genetic line x treatment and gender x treatment interactions were detected for IMF. The LM of Berkshire pigs fed ractopamine had lower (P < 0.05) IMF than Berkshires fed the control diet, with no interaction in the other lines. Purebred barrows (Berkshire and Duroc) had greater (P < 0.001 and P < 0.05, respectively) IMF than their respective purebred gilts, with no gender difference in IMF in the high-lean line. Results from the present study indicate that feeding ractopamine does not affect most muscle quality and palatability characteristics. However, the genetic line x treatment interaction for loin IMF suggests that feeding ractopamine might reduce IMF within the loin muscle of genetic lines that have a propensity to produce greater levels of IMF.

Functional replacement of the essential ESS1 in yeast by the plant parvulin DlPar13.

A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins.

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. Here we show that the major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs.

A microendoscopic test card for ophthalmic endoscopes.

Ophthalmic endoscopes allow examination of intraocular structures when traditional surgical microscopic visualization is limited or impossible. Periodic evaluation of the optical precision of these instruments is essential to ensure optimal intraoperative performance. A test card has been developed to aid in the calibration of ophthalmic endoscopes. Its 5 test targets assess white balance and fiberoptic bundle integrity, focus and resolution, orientation, color fidelity, and angle of view.

Modular structure of the trigger factor required for high activity in protein folding.

The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans isomerase (PPIase) which catalyzes proline-limited protein folding extremely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments which encompass individual domains or combinations of the middle FKBP-like domain (M) with the N-terminal (N) and C-terminal (C) regions, respectively. These fragments appear to be stably folded. They show ordered structure and cooperative urea-induced unfolding transitions, and the far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the native trigger factor shows a modular structure, which is composed of three fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activity in protein folding could not be restored by fusing alternatively the N or the C-terminal regions to the catalytic domain (in NM and MC constructs, respectively). Surprisingly, the high folding activity of the intact trigger factor has been regained partially by functional complementation of the overlapping NM and MC constructs.

Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12.

A low degree of amino acid sequence similarity to FK506-binding proteins (FKBPs) has been obtained for the peptidyl prolyl cis/trans isomerase (PPIase) domain of E. coli trigger factor (TF) that was thought to be significant with regard to the enzymatic properties of the bacterial enzyme. We examined whether the alteration of a negatively charged side-chain at position 37 (FKBP numbering) and a phenylalanine at position 99, both highly conserved through both types of enzymes, leads to parallel effects on the catalytic activity of both FKBP12 and TF-PPIase domain in a series of tetrapeptide substrates with different P1 subsites. For the latter enzyme, substitution of Glu178 by Val or Lys, which aligns to Asp37 in human FKBP12, enhanced the PPIase activity, whereas a strongly decreased enzymatic activity was determined for the Asp37Leu and Asp37Val variants of FKBP12. Regardless of the P1 subsite of the substrate used for the assay, mutation of Phe233Tyr generated a protein variant of the TF-PPIase domain with about 1% of the wild type PPIase activity. Dependent on the substrate nature, a moderate decrease as well as a 4.8-fold increase in k(cat)/K(M) could be determined for the corresponding Phe99Tyr FKBP12 variant. Neither of the mutations of the TF-PPIase domain was able to implant FK506 inhibition found as a major characteristic of the FKBP family of PPIases.

Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding.

The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. In its enzymatic mechanism the trigger factor follows the Michaelis-Menten equation. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 microM. In contrast, the catalytic constant kcat is small and shows a value of 1.3 s(-1) at 15 degrees C. An unfolded protein inhibits the trigger factor in a competitive fashion. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. Together, this suggests that the good substrate binding, i.e. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding.

Isolation and amino acid sequence of a new 22-kDa FKBP-like peptidyl-prolyl cis/trans-isomerase of Escherichia coli. Similarity to Mip-like proteins of pathogenic bacteria.

We identified a periplasmic peptidyl-prolyl cis/trans-isomerase (PPIase) of the (FK506-binding protein (FKBP) type in Escherichia coli (FK506 represents a natural peptidomacrolide containing an acylated pipecolic acid residue). After purification to homogeneity, its complete amino acid sequence was determined by a combination of Edman degradation and electrospray mass spectrometry of the authentic protein and peptides generated by proteolysis. The molecular mass calculated from the amino acid sequence of the protein was 22,085.53 Da, which corresponded perfectly with the value of 22,084 +/- 1.47 Da as determined by mass spectrometry. The corresponding gene was cloned and analyzed, and Southern blot experiments revealed the existence of similar genes in various Gram-negative bacteria. The amino acid sequence of the novel FKBP22 shows similarity to Mip (macrophage infectivity potentiator)-like proteins produced by a number of pathogenic bacteria. However, FKBP22 is inhibited more strongly by FK506 than are other Mip-homologues, as indicated by the Ki value of 25 nM. The subsite specificity regarding the P1 position of the substrate resembles that for Mip-FKBP25 from Legionella pneumophila. The mature FKBP22 enzyme of 205 amino acids exists as a dimer in solution.

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity.

The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cis/trans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatically active fragment could be isolated after proteolysis by subtilisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni2+-NTA column. Subsequent digestion with subtilisin and anion-exchange chromatography yielded a TF fragment encompassing amino acids Gln-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 microM(-1) s(-1) could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrolide FK506.

A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.