Slow Migrating Band Research Articles

TRNATyr has an unusually short half-life in Trypanosoma brucei.

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.

Manganese superoxide dismutase in Meloidogyne incognita isolates selected for virulence on Mi-1-carrying tomato: gene expression and enzyme activity

Root knot nematodes (Meloidogynespp.) are important pests of a wide range of crops, including tomato. Resistance of tomato to root-knot nematodes is conferred by the single dominant geneMi-1, which currently is present in all commercially available resistant tomato cultivars. However, several resistance-breaking populations are being collected worldwide. Two isolates coming from the same standard population ofMeloidogyne incognita, one selected for virulence againstMi-1(SM1) and the other left avirulent (avr1), have been used in this study. qRT-PCR was used to detect transcript levels of the manganese superoxide dismutase (Mn-SOD) gene from (a)virulent pre-parasitic second-stage juveniles (J2). Over-expression of theMn-SODgene was found in the virulent isolate compared with the avirulent counterpart. The enzyme activity of membrane-bound mitochondrial Mn-SOD was assayed in J2 and adult females as the fraction of total SOD activity insensitive to hydrogen peroxide (H2O2). J2 fromSM1showed about a two-fold higher enzyme activity than J2 fromavr1; conversely, no difference was found when adult females were tested. Proteins of J2 extracts were separated by n-PAGE on special mini-gels and stained for SOD. One slow migrating and three fast migrating bands were stained. SOD activity of the slow migrating band was H2O2-insensitive and enriched by treatment with the detergent Triton X-100. J2 survival was monitored in suspensions provided or not provided with the cell oxygen radical generator paraquat, at high concentration. Virulent J2 responded to paraquat treatment by increasing life extension with respect to control conditions; by contrast, avirulent J2 suffered major mortality in the presence of paraquat.Mn-SODgene expression is discussed in relation to nematode fitness in oxidative stresses andvirphenotype.

Identification of a phosphorylation site in cyclobutane pyrimidine dimer photolyase of rice

Cyclobutane pyrimidine dimer (CPD) photolyase monomerises ultraviolet (UV) radiation-induced CPDs present in DNA, using energy from UVA and visible light. In plants, CPD photolyase activity is a crucial factor for determining UVB sensitivity. We previously demonstrated that native rice CPD photolyase is phosphorylated. To determine the phosphorylation site(s), the phosphorylation status of CPD photolyase was analyzed in rice varieties that have amino acid alterations at the potential phosphorylation sites. In wild-rice species, CPD photolyase was phosphorylated. In Poaceae species, CPD photolyase was phosphorylated in wheat but not in maize. Mutant CPD photolyase proteins, in which these putative phosphorylated residues were replaced with alanine residues, were synthesized using an insect cell-free translation system. A slow-migrating band disappeared when the serine residue at position 7 was mutated. A phospho-specific antibody was generated to determine whether this residue is phosphorylated in CPD photolyase. Only the slow-migrating band of native rice CPD photolyase was detected using this antibody, indicating that the serine residue at position 7 is a phosphorylation site in native rice CPD photolyase.

Reconstitution of Yeast Silent Chromatin: Multiple Contact Sites and O-AADPR Binding Load SIR Complexes onto Nucleosomes In Vitro

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Antioxidant enzymes in (a)virulent populations of root-knot nematodes

AbstractAssays of antioxidant enzymes, including catalase, peroxidase and superoxide dismutase (SOD), have been carried out on extracts of females and second-stage juveniles (J2) of a pair of Meloidogyne incognita isolates, one virulent and one avirulent, selected on tomato, and an avirulent field population of M. incognita. Catalase and SOD activity were found to be higher in extracts of the virulent isolate SM1 when compared with the avirulent counterparts. Peroxidase activity, assayed with o-dianisidine as the substrate, was enhanced in SM1 J2 compared with the avirulent avr1 J2. Catalase isozymes were separated by isoelectrofocusing into a very acidic and a basic isoform; this latter isoform was found to be responsible for the enhancement of catalase activity in virulent populations. SOD isozyme electrophoresis patterns (IEP) of root-knot nematodes, obtained by native PAGE, showed the presence of slow- and fast-migrating bands. SOD IEP of virulent females contained a slow-migrating band with a relative mobility (Rm) on the gels slightly higher (0.52) than the corresponding band from avirulent populations (0.50). This change was confirmed with native PAGE gels loaded with extracts from J2. To check how widespread this change is in field populations of RKN, a survey of SOD IEP from 12 RKN field (a)virulent populations was carried out. The specificity of the 0.52 Rm band for virulent populations was confirmed. Separation by native PAGE of peroxidases, stained either by o-dianisidine or diamino-benzidine, showed two isoforms with no apparent differences between the populations tested.

Inhibitors of Protein Geranylgeranyltransferase I and Rab Geranylgeranyltransferase Identified from a Library of Allenoate-derived Compounds

Protein geranylgeranylation is critical for the function of a number of proteins such as RhoA, Rac, and Rab. Protein geranylgeranyltransferase I (GGTase-I) and Rab geranylgeranyltransferase (RabGGTase) catalyze these modifications. In this work, we first describe the identification and characterization of small molecule inhibitors of GGTase-I (GGTI) with two novel scaffolds from a library consisting of allenoate-derived compounds. These compounds exhibit specific inhibition of GGTase-I and act by competing with a substrate protein. Derivatization of a carboxylic acid emanating from the core ring of one of the GGTI compounds dramatically improves their cellular activity. The improved GGTI compounds inhibit proliferation of a variety of human cancer cell lines and cause G(1) cell cycle arrest and induction of p21(CIP1/WAF1). We also report the identification of novel small molecule inhibitors of RabGGTase. These compounds were identified first by screening our GGTI compounds for those that also exhibited RabGGTase inhibition. This led to the discovery of a common structural feature for RabGGTase inhibitors: the presence of a characteristic six-atom aliphatic tail attached to the penta-substituted pyrrolidine core. Further screening led to the identification of compounds with preferential inhibition of RabGGTase. These compounds inhibit RabGGTase activity by competing with the substrate protein. These novel compounds may provide valuable reagents to study protein geranylgeranylation.

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and three l-glycero-d-manno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.

A Functional Enhancer of Keratin14 Is a Direct Transcriptional Target of ΔNp63

Keratin14 (K14) is a prototypic marker of dividing basal keratinocytes where its gene is transcribed at high levels. Transcriptional regulation of K14 is governed by an evolutionarily conserved functional enhancer marked by DNase 1 hypersensitive sites present upstream of the gene. This enhancer is sufficient to confer epidermal-specific gene expression, which is mediated in part by binding of members of activator protein-2 (AP)-2, AP-1, Ets, and Sp1 families of transcription factors. Here we provide evidence that a keratinocyte-specific nuclear protein identified as deltaNp63 binds to a conserved motif within this enhancer. Interestingly, the selective expression profile of deltaNp63 in various cell lines correlates with both the nuclear complex and the expression of K14. Biochemical studies reveal that deltaNp63 can bind to a specific DNA sequence present in the K14 enhancer and this binding leads to transactivation. In addition, chromatin immunoprecipitation experiments with deltaNp63-specific antibodies demonstrate that the enhancer is occupied by deltaNp63 in cultured keratinocytes and in mouse skin epidermal cells in vivo. Finally, we show that ectopic expression of either p63 isoform (deltaN or TA) can induce de novo expression of K14. These studies provide a potential mechanism by which deltaNp63 directly governs the expression of K14 in a keratinocyte-specific manner.

Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within singleFrankiastrains

Detection of polymorphisms in intergenic transcribed spacer (ITS) 16S-23S rRNA within single Frankia strains. Polymorphisms in the 16S-23S rRNA ITS were investigated in single-colony subcultures of seven Frankia isolates. Multiple ITS-polymerase chain reaction (PCR) bands were detected solely in isolates BMG5.5 and BMG5.11. The slow-migrating bands in the ITS-PCR agarose gel electrophoresis profiles of the isolates were revealed to be heteroduplexes on the basis of their migration shift in different electrophoretic matrices, southern hybridization and the single-strand DNA mung bean endonuclease digestion. Laser-scanned capillary electrophoresis detected two ITS-PCR fragments differing in length by three and six nucleotide insertions/deletions in strains BMG5.5 and BMG5.11, respectively. Sequence analysis of the cloned ITS showed that in strain BMG5.5 the two ITS differed by the presence of three to four copies of the 3-bp tandem repeat 5'-TGG-3'. In strain BMG5.11, the two ITS differed by the presence of two to three copies of the 6-bp tandem repeat 5'-CTTGGG-3'. We demonstrate the occurrence of ITS 16S-23S rRNa polymorphisms within single Frankia strains. We reported the occurrence of ITS 16S-23S rRNA polymorphisms within single Frankia strains from Elaeagnus host group recognized as the more flexible strains within Frankia genus. Furthermore, we underscored the applied interest of strains BMG5.11 and BMG5.5 in future ecological studies using ITS 16S-23S rRNA as molecular marker.

Site-specific Phosphorylation and Caspase Cleavage Differentially Impact Tau-Microtubule Interactions and Tau Aggregation

The microtubule-associated protein tau is hyperphosphorylated and forms neurofibrillary tangles in Alzheimer disease. Additionally caspase-cleaved tau is present in Alzheimer disease brains co-localized with fibrillar tau pathologies. To further understand the role of site-specific phosphorylation and caspase cleavage of tau in regulating its function, constructs of full-length tau (T4) or tau truncated at Asp421 (T4C3) to mimic caspase-3 cleavage with and without site-directed mutations that mimic phosphorylation at Thr231/Ser235, Ser396/Ser404, or at all four sites (Thr231/Ser235/Ser396/Ser404) were made and expressed in cells. Pseudophosphorylation of T4, but not T4C3, at either Thr231/Ser235 or Ser396/Ser404 increased its phosphorylation at Ser262 and Ser199. Pseudophosphorylation at Thr231/Ser235 impaired the microtubule binding of both T4 and T4C3. In contrast, pseudophosphorylation at Ser396/Ser404 only affected microtubule binding of T4C3 but did make T4 less soluble and more aggregated, which is consistent with the previous finding (Abraha, A., Ghoshal, N., Gamblin, T. C., Cryns, V., Berry, R. W., Kuret, J., and Binder, L. I. (2000) J. Cell Sci. 113, 3737-3745) that pseudophosphorylation at Ser396/Ser404 enhances tau polymerization in vitro. In situ T4C3 was more prevalent in the cytoskeletal and microtubule-associated fractions compared with T4, whereas purified recombinant T4 bound microtubules with higher affinity than did T4C3 in an in vitro assay. These data indicate the importance of cellular factors in regulating tau-microtubule interactions and that, in the cells, phosphorylation of T4 might impair its microtubule binding ability more than caspase cleavage. Treatment of cells with nocodazole revealed that pseudophosphorylation of T4 at both Thr231/Ser235 and Ser396/Ser404 diminished the ability of tau to protect against microtubule depolymerization, whereas with T4C3 only pseudophosphorylation at Ser396/Ser404 attenuated the ability of tau to stabilize the microtubules. These results show that site-specific phosphorylation and caspase cleavage of tau differentially affect the ability of tau to bind and stabilize microtubules and facilitate tau self-association.

Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondria-rich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

Histone H1 is phosphorylated in non-replicating and infective forms of Trypanosoma cruzi

The nuclear structure changes during the differentiation from growing to infective stages of Trypanosoma cruzi. As histone modifications have been correlated with structural and functional changes of chromatin, we investigated whether histones in T. cruzi are modified during the life cycle of this protozoan parasite. We found that histone H1 isolated from proliferating forms (epimastigotes) and from differentiated/infective forms (trypomastigotes) have a distinct migrating pattern in Triton–acetic acid–urea gel electrophoresis. While epimastigotes contain predominantly a fast migrating form, a slow migrating band is prominent in trypomastigotes. By metabolically labeling the cells with radioactive phosphate, we demonstrated that the slow migrating histone H1 band is phosphorylated, and that after alkaline phosphatase treatment, it migrates as the fast form. Parasites arrested at the onset of the S phase of the cell cycle with hydroxyurea (HU) also predominantly have the phosphorylated form of histone H1, suggesting that phosphorylation occurs in non-replicating stages of T. cruzi. We also found that the phosphorylated histone H1 is more weakly associated with the chromatin, being preferentially released at 150 mM NaCl. Therefore, histone H1 phosphorylation varies during the life cycle of T. cruzi, and might be related to changes in the chromatin structure.

Duality Monomer–Dimer of the Pheromone-Binding Protein from Bombyx mori

The analysis of a recombinant pheromone-binding protein from the silkworm moth, Bombyx mori, by native gel electrophoresis with Coomassie staining showed one single band with a molecular mass consistent with a monomer. A slow migrating band, detected in the recombinant and native samples by a polyclonal antibody, was indistinguishable from the monomer in the mass spectrum fragmentation pattern and chromatographic behavior. Flow injection analyses of the protein by mass spectrometry in the negative mode showed fragments of a dimer. The dimeric form was also supported by estimation of the molecular mass by gel filtration at basic pH. A cross-linked dimer coeluted with the noncovalent dimer on a gel filtration column. The molecular mass of the protein changed in a pH-dependent way with a dramatic transition from dimer to monomer between pH 6 and 4.5. A low pH induced not only dissociation of the dimer, but also a conformational change in the protein. In marked contrast to denaturation with guanidinium chloride, the emission maxima of tryptophan was not significantly changed at low pH. BmPBP is thus a dimer at slightly acid, neutral, and basic pH, which dissociates and then undergoes conformational change at low pH.

The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila.

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.

Thermal stability of soluble malate dehydrogenase isozymes of subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes

Electrophoretic thermostability tests of soluble malate dehydrogenases (sMDH) isozymes in tissue extracts of 21 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes showed three distinct results. The first, characterized by thermal stability of the slowest-migrating band or A-isoform, was detected in 52% of all species. The second, exhibited in 29% of the species analyzed, had a bidirectionally divergent pattern of their sMDH locus expression, and was characterized by a nondivergent thermostability pattern of both sMDH-A* and B*. In the third category, obtained in 19% of the species studied (the four Siluriformes species), thermostability of the fastest-migrating bands, or B-isoforms, was observed. Comparison of the effects of habitat temperature on the activity of paralogous and orthologous isoforms in tissue extracts of two of these species with different thermostability properties (Leporinus friderici - thermostable sMDH-A*, and Pimelodus maculatus - reverse thermostability properties or reverse electrophoretic pattern), collected during winter and summer months, showed that A and B subunits were present at different quantitative levels and their activities were nearly season independent. Differences in susceptibility to temperature (50°C) of both sMDH loci from tissue extracts of these species were found. In P. maculatus, these susceptibilities helped strengthen one of the hypotheses: the reverse thermostability pattern, where the fastest-migrating band or the B-isoform was the thermostable sMDH. Thus, temperature differences among orthologous homologues of sMDH seem to have occurred in these acclimatized species, where the fastest-migrating band, usually muscle specific and thermolabile in most teleosts, appeared in P. maculatus as the thermostable isoform.

The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

Presence of an unusually high concentration of an ubiquitinated histone-like protein in Trypanosoma cruzi.

The conjugation of ubiquitin to histones H2A and H2B has been established in higher eukaryotes and has been related to changes in chromatin organization. In Trypanosoma cruzi, no condensation of chromatin occurs during mitosis. In order to determine the presence of histone ubiquitination in T. cruzi epimastigotes, histones were extracted from chromatin and analyzed by three electrophoretic systems: acid-urea, triton-acid-urea and sodium-dodecyl-sulphate polyacrylamide gel. The immunochemical detection of ubiquitin-histone conjugates by Western blotting showed a strong reaction with a slow migrating band of M(r) 19 kDa. The high percentage of ubiquitin-histone conjugates present in T. cruzi chromatin may be related to the inability of this parasite to condense chromatin into a 30 nm fiber.

Isozyme and Randomly Amplified Polymorphic DNA (RAPD) Analyses of Cherokee Rose and Its Putative Hybrids `Silver Moon' and `Anemone'

Two banding patterns were revealed by phosphoglucomutase (PGM) isozyme analysis of 24 accessions of Cherokee rose (Rosa laevigata Michx.) from eight southeastern states, based on the presence (in 5 accessions) or absence (in 19 accessions) of an additional slow-migrating band. RAPD analysis of these accessions showed a corresponding division into the same two groups determined by PGM analysis, except for two accessions with unique RAPD phenotypes. Field-grown accessions showed distinguishing morphological characters corresponding to the groupings from the isozyme and RAPD analyses. Those in the predominant isozyme and RAPD groups, as well as the two with unique RAPD phenotypes, exhibited smooth lateral stems, while those in both nonpredominant groups exhibited markedly bristly laterals. These results suggest that the 24 accessions are ramets of two major clones with one clone predominating and that, contrary to long-standing belief, the Cherokee rose has not naturalized by reseeding in the southeast. PGM and RAPD analyses of putative Cherokee rose hybrids `Anemone' and `Silver Moon' showed that `Anemone' is likely to be such a hybrid but that `Silver Moon' is not. Historical records revealed that widespread vegetative propagation of the Cherokee rose was initiated in 1820-21 and that L. Wiesener, not J.C. Schmidt, was the originator of `Anemone'.

Plasmin-mediated proteolysis of osteocalcin.

Plasmin cleaves osteocalcin at a site within its carboxyl end, thus creating an N-midterminal 1-43 and a short C-terminal 44-49 peptides. The products of the cleavage were identified by matrix assisted laser desorption ionization time of flight mass spectrophotometry and by reversed phase high performance liquid chromatography followed by N-terminal sequence determination. When separated by sodium dodecyl sulfide-polyacrylamide gel electrophoresis in the presence of reducing agents, large (LF; N-midterminal) and a small molecular weight (SF; C-terminal) fragments can be identified. The major cleavage site involves arg43-arg44 amino acid residues, and the resulting 44-49 C-terminal fragment appears as a slow migrating band on native gels (SFnat). Elevated levels of calcium ion inhibit the plasmin-mediated lysis of osteocalcin. Plasmin-mediated cleavage of osteocalcin occurs both in solution and when bound to hydroxyapatite. Both osteocalcin cleavage products detach from the hydroxyapatite substrate. Diisopropyl fluorophosphate-inhibited plasmin does not displace osteocalcin from the hydroxyapatite surface. Previously, the C-terminal pentapeptide has been shown to be chemotactic for bone cells while bone particles lacking osteocalcin were resistant to bone resorption. We therefore hypothesize that the plasmin-mediated digestion of free and hydroxyapatite-bound osteocalcin could play a role in the regulation of bone remodeling.

Effect of electrical stimulation on postmortem titin, nebulin, desmin, and troponin-T degradation and ultrastructural changes in bovine longissimus muscle.

Electrical stimulation (ES) of bovine carcasses is usually done to increase tenderness and has been hypothesized to increase the activity of proteolytic enzymes that may degrade structural proteins in muscle cells and cause fractures and breaks in muscle fibers, thus enhancing meat tenderness. Our objective was to compare postmortem (PM) changes in the muscle proteins, titin, nebulin, alpha-actinin, desmin, and troponin-T and in myofibrillar structure in nonstimulated (NS) and ES bovine skeletal muscle. One side of eight beef carcasses was stimulated within 1 h of death, and the other side was the NS control. Myofibrils for SDS-PAGE and samples for transmission electron microscopy were prepared from the longissimus muscle at 0, 1, 3, 7, 14, and 28 d PM. In SDS-PAGE, titin migrated as three bands in both NS and ES 0-d samples. The slowest migrating band, T1 (intact titin), decreased slightly faster in ES samples from five animals. The fastest migrating band, T2 (degraded titin), increased in amount through 3 d and was still present at 28 d. A titin monoclonal antibody (mAb) identified a large family of degradation products that migrated faster than myosin heavy chains and that was more heavily labeled in Western blots of ES samples than in NS samples. In SDS-PAGE of NS samples, intact nebulin disappeared by 3 d in three animals, by 7 d in four animals, and by 14 d in one animal, but in ES samples the nebulin band was absent by 3 d in three animals and by 7 d in five animals. SDS-PAGE showed that the amount of intact desmin decreased slightly sooner in two ES samples and was absent earlier in one ES sample than in the corresponding NS control samples. Blots labeled with a polyclonal antibody to desmin showed that a more heavily labeled 38-kDa desmin degradation product was present in ES than in NS samples. Postmortem degradation of alpha-actinin was not detected. Contraction node (CN) formation, stretching of conjoined sarcomeres adjacent to the nodes, increased frequency of I-band fractures and accelerated appearance of wide I-band fractures adjacent to the Z-line, and, in some animals, slightly accelerated degradation of titin, nebulin, and troponin-T were characteristics of ES muscle.