Thesis: Ph. D., Massachusetts Institute of Technology, Department of Nutrition and Food Science, 1976 / Vita. / Includes bibliographical references. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Nutrition and Food Science

Surimi is a seafood product which is used to manufacture restructured products such as artificial crab and lobster. Surimi is produced from fish fillets by washing to remove sarcoplasmic proteins and increase the concentration of myofibrillar proteins, and mixing with cryoprotectants. A valuable attribute of surimi is its ability to form an elastic gel, the gel network being formed by the myofibrillar proteins of fish muscle. It is generally accepted that the quality of surimi gels is influenced by the activity of endogenous protease which acts on the myofibrillar proteins. The proteases in Pacific whiting surimi (Merluccius productus) are particularly problematic due to their high catalytic activity on muscle myosin. The addition of whey protein concentrate (WPC) to Pacific whiting surimi has been shown to enhance the gel strength of the corresponding products produced from this surimi. The mechanism through which WPC enhances the gel strength of Pacific whiting surimi has not been determined, but it has been suggested that WPC acts to inhibit surimi autoproteolysis. The objective of this study was to determine whether the incorporation of WPC into Pacific whiting surimi inhibits autoproteolysis and/or protects the myosin fraction from proteolytic degradation. The effect of supplementing surimi with WPC, beef plasma protein (BPP) and bovine serum albumin (BSA) on its apparent autoproteolysis activity was determined. Three WPC preparations were tested, WPC 34, 34% protein; WPC 80, 80% protein; and WPC 95, 95% protein. Each of the additives was incorporated at the 1, 2, 3 or 4% level. Proteolysis of surimi and supplemented surimi samples was allowed to occur at 55°C. Proteolytic reaction mixtures were terminated by the addition of trichloroacetic acid (TCA). Proteolytic activity was estimated by measuring the difference in TCA-soluble peptides present in reaction mixtures of paired (identical) samples, one having been incubated at 55°C while the paired sample was kept on ice. Peptides were quantified by the bicinchoninic acid, Lowry, dye-binding and trinitrobenzenesulfonic acid methods. Results based on the different peptide assays were compared in order to asses the reliance of results on specific assay methods. BPP was found to have the most inhibitory activity in the autoproteolysis assays, followed by the WPC preparations and then BSA. Autoproteolysis was completely inhibited by the incorporation of 1% BPP, 3% WPC 80 and 2% WPC 95. The extent of inhibition by the WPC preparations was related to their protein content, the higher the protein content the greater the extent of inhibition per unit weight added to surimi. BSA was not an inhibitor of autoproteolysis under the conditions used in this study. The relative extents of inhibition observed for the different additives were independent of the method used to quantify the soluble peptide products. Each of the additives was also tested for their ability to protect the myosin component of surimi from proteolytic degradation. These experiments were done as described above for the autoproteolysis assays with the exception that following the incubation period a portion of the sample, either surimi or a surimi/additive mixture, was completely solubilized in detergent solution, subjected to SDS-PAGE electrophoresis and visualized by protein staining. In these experiments the additives were incorporated at the 4% level. No apparent degradation of myosin could be detected over a 60 min reaction period for surimi samples that were supplemented with BPP, WPC 80 and WPC 95. In contrast, surimi samples incubated without additive clearly showed a loss of myosin after 15 min reaction period. Some myosin degradation was apparent following the 60 min incubation period for the WPC 34-supplemented surimi. A further experiment was conducted to determine the mechanism through which WPC protects myosin and inhibits autoproteolysis. In this experiment WPC 95 and BPP were separately incubated at 55°C with a crude fish protease preparation, i.e. the reaction mixture approximates that used in the autoproteolysis assays except that it contains no surimi. The results indicate that BPP and WPC 95 behave in a similar manner. However, the results were inconclusive with regard to explaining the additive's mechanism of action. Plausible mechanisms which are consistent with the results are discussed. / Graduation date: 1994

A novel method for isolating fish proteins by shifting pH to high acid or high alkali pH was the focus of the study. Biochemical and physicochemical properties of various pH-treated soluble fish proteins as a function of ionic strength were determined. Effect of ionic strength and various storage conditions on gelation properties and stabilization of fish protein isolate (FPI) were also elucidated. At low ionic strength (IS 10 mM NaCl), the solubility of Pacific whiting (PW) proteins was low between pH 5 and 10, but increased significantly as the pH was shifted to either acidic or alkaline pH. The isoelectric point (pi) shifted toward acidic direction as IS increased to 600 mM. High IS (600 mM NaCl) resulted in protein aggregation at low pH but improved myosin heavy chain (MHC) solubility at pH 6 - 10. Changes in total sulfhydryl (SH) content and surface hydrophobicity (S [subscript o]) were associated with the different molecular weight distributions of the soluble proteins. At pH 4 and IS 10-100 mM, MHC was soluble but degraded. At pH 10, the formation of high MW polymers was observed at IS [greater than or equal to] 150 mM. Gels obtained from FPI prepared at pHl1/IS150 and conventional surimi (CS) were superior to FPI prepared at pH 3 and/or other IS levels. There was no correlation between protein solubility and gel properties of FPI. Gelation mechanisms of acid and alkali-treated FPI were identical under the same IS condition. FPI prepared at pH 3 or 11 could be partly refolded at pH 7. No significant difference in texture was observed between alkali-treated protein isolates (AKPI, pH 11) kept frozen at pH 5.5 and 7.0. Strongest gel was found for AKPI with cryoprotectants (C) and without freeze/thaw (FT) cycles at both pH storage (5C & 7C), while poor gel was obtained from AKPI without cryoprotectants (NC) and with FT (5NC-F & 7NC-F). 5NC-F & 7NC-F demonstrated the lowest S [subscript o] and total SH probably suggesting that proteins were more aggregated as a result of hydrophobic interactions and disulfide bonds. Scanning electron microscope (SEM) revealed the most discontinuity of gels from AKPI without cryoprotectants and with FT and showed less protein stability when stored at pH 5.5 than at neutral pH. Raman spectral analysis demonstrated that refolding of AKPI by pH adjustment to 7.0 was achieved, but not identical to the native protein. CS contained higher α-helix content (~50%) than AKPI (~20-30%). Frozen storage induced a decrease and an increase in the α-helix of CS and AKPI samples, respectively. Alkali-treated proteins were slightly less stable than CS during frozen storage. / Graduation date: 2005

The effects of denervation, non-weight bearing (unloading) or immobilization on hindlimb muscle growth, protein and amino acid metabolism were studied. In the first 3 days after denervation or unloading, atrophy of the soleus was caused by a suppression of protein synthesis and an acceleration of protein degradation. Thereafter, further atrophy, up to 6 days was due to depressed protein synthesis only. The changes in both protein synthesis and degradation in the first three days accounted for 69% and 65%, respectively, of the total loss of protein and mass in 6 days of unloaded or denervated soleus. Over the 6-day period, denervated soleus lost more mass and protein than the unloaded muscle owing to the earlier onset and greater extent of proteolysis. In denervated soleus, both lysosomal and non-lysosomal proteolysis may be enhanced, whereas in the unloaded muscle possibly only non-lysosomal proteolysis was enhanced. In both cases non-lysosomal proteolysis may be mediated by Ca²⁺-activated neutral protease, partially as a result of Ca²⁺ release from sarcoplasmic reticulum. Possibly due to the lack of lysosomal proteolysis, the insulin receptor did not show apparent increased turnover with unloading, as suggested by increased insulin sensitivity of in vitro protein turnover in the unloaded soleus. In contrast, denervated soleus showed a normal response to insulin for in vitro protein turnover. These findings suggested a mechanistic difference of unloading and denervation atrophy of soleus. A decreased ratio of glutamine/glutamate in fresh muscle suggested that the synthesis of glutamine in soleus may be diminished by denervation just as by unloading. This diminution of glutamine synthesis was probably due to reduced availability of ammonia, as evidenced by the slow disappearance of ATP in incubated denervated soleus. Similiar to unloading, denervation led to a decrease in aspartate concentration. This decreased concentration apparently resulted in decreased rather than increased utilization of aspartate. Effects of stretch on unloaded soleus were particularly pronounced in the first two days. Thereafter, in the stretched, unloaded soleus protein degradation increased to nearly the same extent as did protein synthesis. Hence after two days, stretch seems to lose its effectiveness in mitigating the effects of unloading so that it may not be an adequate preventive measure of muscle wasting under non-weight bearing condition.

In vivo protein turnover was measured during the last 4 days of flight muscle development in tobacco hornworm pupa. Linear synthesis rates were measured up to 2 hours after injection of 30 μmol (3H) phenylalanine. Since the results with this technique did not differ from another established method, the large bolus injection of phenylalanine did not affect protein synthesis. The former method is advantageous because only a single time point is required. Flight muscle growth and protein synthesis decreased in parallel between 100 and 24 hours. During this time free phenylalanine turnover decreased, and the total pool diminished, indicating that this pool could be a major sink for muscle protein synthesis. Proteolysis was rapid even in the growing muscle. 20-Hydroxyecdysone increased muscle growth at certain times by inhibiting proteolysis. Protein synthesis either decreased or was unchanged after injection of the hormone. Therefore ecdysteroids may play a role in controlling growth of the dorsolongitudinal flight muscle during adult development, especially by retarding proteolysis.

Cells contain an elaborate cytoskeleton which plays a major role in a variety of cellular functions including: maintenance of cell shape and dimension, providing mechanical strength, cell motility, cytokinesis during mitosis and meiosis and intracellular transport. The cell cytoskeleton is made up of three types of protein filaments: the microtubules, the intermediate filaments and the actin cytoskeleton. These components interact with each other to allow the cell to function correctly. When functioning incorrectly, disruptions to many cellular pathway have been observed with mutations in various cytoskeletal proteins causing an assortment of human disease phenotypes. Characterization of these filament systems in different cell types is essential to the understanding of basic cellular processes and disease causation. The studies in this thesis are concerned with examining specific cytoskeletal tropomyosin-defined actin filament systems in skeletal muscle. The diversity of the actin filament system relies, in part, on the family of actin binding proteins, the tropomyosins (Tms). There are in excess of forty Tm isoforms found in mammals which are derived from four genes: α, β, γ and δTm. The role of the musclespecific Tms in striated muscle is well understood, with sarcomeric Tm isoforms functioning as part of the thin filament where it regulates actin-myosin interactions and hence muscle contraction. However, relatively little known about the roles of the many cytoskeletal Tm isoforms. Cytoskeletal Tms have been shown to compartmentalise to form functionally distinct filaments in a range of cell types including neurons (Bryce et al., 2003), fibroblasts (Percival et al., 2000) and epithelial cells (Dalby-Payne et al., 2003). Recently it has been shown that cytoskeletal Tm, Tm5NM1 defines a cytoskeletal structure in skeletal muscle called the Z-line associated cytoskeleton (Z-LAC) (Kee et al., 2004).The disruption of this structure by over-expression of an exogenous Tm in transgenic mice results in a muscular dystrophy phenotype, indicating that the Z-LAC plays an important role in maintenance of muscle structure (Kee et al., 2004). In this study, specific cytoskeletal Tms are further investigated in the context of skeletal muscle. Here, we examine the expression, localisation and potential function of cytoskeletal Tm isoforms, focussing on Tm4 (derived from the δ- gene) and Tm5NM1 (derived from the γ-gene). By western blotting and immuno-staining mouse skeletal muscle, we show that cytoskeletal Tms are expressed in a range of muscles and define separate populations of filaments. These filaments are found in association with a number of muscle structures including the myotendinous junction, neuromuscular junction, the sarcolemma, the t-tubules and the sarcoplasmic reticulum. Of particular interest, Tm4 and Tm5NM1 define cytoskeletal elements in association with the saroplasmic reticulum and T-tubules, respectively, with a separation of less than 90 nm between distinct filamentous populations. The segregation of Tm isoforms indicates a role for Tms in the specification of actin filament function at these cellular regions. Examination of muscle during development, regeneration and disease revealed that Tm4 defines a novel cytoskeletal filament system that is orientated perpendicular to the sarcomeric apparatus. Tm4 is up-regulated in both muscular dystrophy and nemaline myopathy and also during induced regeneration and focal repair in mouse muscle. Transition of the Tm4-defined filaments from a predominsnatly longitudinal to a predominantly Z-LAC orientation is observed during the course of muscle regeneration. This study shows that Tm4 is a marker of regeneration and repair, in response to disease, injury and stress in skeletal muscle. Analysis of Tm5NM1 over-expressing (Tm5/52) and null (9d89) mice revealed that compensation between Tm genes does not occur in skeletal muscle. We found that the levels of cytoskeletal Tms derived from the δ-gene are not altered to compensate for the loss or gain of Tm5NM1 and that the localisation of Tm4 is unchanged in skeletal muscle of these mice. Also, excess Tm5NM1 is sorted correctly, localising to the ZLAC. This data correlates with evidence from previous investigations which indicates that Tm isoforms are not redundant and are functionally distinct (Gunning et al., 2005). Transgenic and null mice have also allowed the further elucidation of cytoskeletal Tm function in skeletal muscle. Analyses of these mice suggest a role for Tm5NM1 in glucose regulation in both skeletal muscle and adipose tissue. Tm5NM1 is found to colocalise with members of the glucose transport p fibres and analysis of both transgenic and null mice has shown an alteration to glucose uptake in adipose tissue. Taken together these data indicate that Tm5NM1 may play a role in the translocation of the glucose transport molecule GLUT4. In addition to this Tm5NM1 may play a role in adipose tissue regulation, since over-expressing mice found to have increased white adipose tissue and an up-regulation of a transcriptional regulator of fat-cell formation, PPAR-γ. / Doctor of Philosophy (PhD)

The first goal of this study was to understand the role of u-calpain in skeletal muscle protein degradation in cultured muscle cells. Several strategies were developed to down-regulate endogenous u-calpain activity and m-calpain activity in rat myotubes. These included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain (DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS, i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin degradation (an index of calpain activity). Our data supported the contention that u-calpain contributes significantly to total protein degradation in myotubes. Specifically, over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24 hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of antisense u-CL and the 30K subunit reduced total protein degradation significantly at the 24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K) did not affect (P>0.05) the total protein degradation. In addition to this we determined that desmin was a calpain substrate and that calpain could not degrade tropomyosin. The second goal of this study was to evaluate the relationships among u- and m-calpain and the 30KD subunit. The rationale for this study was that our earlier work indicated coordinated regulation of the calpain subunits. Our data demonstrated for the first time that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are coordinately regulated, respectively. However, the expression of u-calpain did not affect the expression of m-calpain The third goal of this study was to develop a skeletal muscle-specific inducible expression system that may be used in transgenic animal research. A skeletal muscle a-actin promoter was used to replace the cytomegalovirus immediate-early promoter (pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity assay indicated that the skeletal muscle-specific expression system functioned in myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had 15-fold higher B-gal activity than the control cells. / Graduation date: 2002

Little is known about protein profiles in slow-twitch (MHC I) and fast-twitch (MHC IIa and MHC IIx) human skeletal muscle fibers. Therefore we developed a method of assessing fiber type specific protein content across the continuum of human skeletal muscle fiber types. The method presented here combines the advantages of SDS-PAGE for fiber typing with the common Western Blot (WB) technique. Individual vastus lateralis muscle fibers (n = 264) were isolated and clipped into two portions, one for fiber-typing and one for protein identification. Following fiber type determination, WB destined fiber segments were combined into fiber type specific pools (20 fibers/pool) and assessed for GAPDH, actin, Citrate Synthase, and total p38 content. GAPDH expression was 69%, 92%, 159%, and 200% more abundant in MHC I/IIa, MHC IIa, MHC IIa/IIx, and MHC IIx pools when compared to MHC I, respectively. Inversely, Citrate synthase content was 526%, 497%, 316%, and 47% more abundant in MHC I, MHC I/IIa, MHC IIa, and MHC IIa/IIx when compared to MHC IIx, respectively. Similar to GAPDH, total p38 expression was 67% greater in MHC IIa versus MHC I fibers. These data establish a novel application of WB combined with SDS-PAGE for fiber type specific protein analysis in human skeletal muscle. These initial results show content of particular proteins exist in a hierarchal fashion throughout the continuum of human skeletal muscle fiber types. Application of these methods will enhance our understanding of skeletal muscle health profiles among physically active and clinically based populations. / Access to thesis permanently restricted to Ball State community only / School of Physical Education, Sport, and Exercise Science

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains x, 152 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 173-178).

published_or_final_version / Dentistry / Master / Master of Orthodontics