Call number: LD2668 .T4 1953 B65 / Master of Science

The first goal of this study was to understand the role of calpains in skeletal muscle protein degradation in cultured cells. We have developed a genetic approach to inhibit endogenous calpain activity through over-expressing dominant negative m-calpain (DN), antisense m-calpain (AS) and calpastatin inhibitory domain (CID). We observed that, under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain through DN-m-calpain over-expression caused a 30% inhibition of total protein degradation whereas CID over-expression reduced degradation by 63%. These constructs did not significantly affect degradation in the presence of serum. These data indicate that calpains participate in the accelerated degradation associated with serum withdrawal. Inhibition of calpain also stabilized nebulin, a major structural protein of the sarcomere. These observations indicate that calpains play significant roles in muscle protein turnover. Finally, over-expression of antisense m-calpain caused a transient reduction in m-calpain concentration after which normal m-calpain concentration was quickly re-established. These observations indicate that m-calpain is a short half-life protein in muscle cells. The second goal of this study is to investigate the role of calpain in the mediation of PARP protein level in differentiating myoblasts. Poly(ADP-ribosyl)ation, catalyzed by PARP, is involved in various physiological events, such as DNA excision repair, DNA recombination, DNA replication, cell differentiation, cell growth and transformation, and apoptosis. A protease participating in PARP turnover could be a significant regulator to the events which PARP is involved. A relationship between apoptosis and myofibrillar protein degradation via a common protease might suggest the basis for muscle wasting and atrophy which characterize in many muscle diseases. We established a genetic approach to inhibit endogenous calpain activity through over-expressing calpastatin inhibitory domain (CID). We observed that (1) inhibition of calpain activity increased PARP concentration when post-confluent myoblasts were cultured with 2% HS medium, an inducer of differentiation and (2) inhibition of calpain activity prevented PARP degradation induced by A23187 and etoposide in differentiating myoblasts. These data demonstrate that calpain is involved in regulation of PARP in cultured cells. / Graduation date: 1998

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

The changes in the level of circulating thyroid hormone, or protein-bound iodine (PBI), in the male Holstein calf and the Wistar Albino rat during a portion of the growth period have been determined. Two levels of nutrition were employed.in each case to determine if the nutritional status exerted an appreciable effect on the measured PBI level. Repetitive measurements of the serum PBI were conducted with each of eight calves in order to assess age changes, while with the rats single determinations were made on animals killed at regular weight intervals. Carcass analysis of the rats, including body fat and water determinations of the individual animals, and protein and ash analysis of a pooled sample from each group, was conducted with the intent of relating age changes of the serum PBI to a measure of "metabolically active tissue". The PBI level of the Holstein calf was found to increase with age in the growth interval considered. The rate of change of the PBI level of the low plane calves appeared to be less than in the high plane animals; the degree of variability was such, however, that a definite effect of nutrition could not be shown. No correlation was found between either the initial PBI level or the level at slaughter and the daily rate of gain. The resting metabolic rate of the calves was determined prior to the removal of a blood sample for PBI analysis. No relationship between the serum PBI level and the metabolic rate existed. In the case of the rats, no correlation was found between the serum PBI level and chronological age, body weight or fat-free body weight. A great deal of variability of the PBI level among animals in the same group was observed. Possible reasons for this variability are discussed. The body composition data of the high plane rats followed accepted trends, with the body water, protein and ash fractions exhibiting differential growth with respect to body weight and fat-free body weight. The composition of the low plane rats was not appreciably different from that of the high plane animals at equal body weights. The realimentation period was characterized by the deposition of large quantities of body fat. The significance of these findings in terms of the results of other investigators is discussed. Various workers have attempted to relate the serum PBI level of a young animal with its potential productive worth. No evidence was obtained in this study of such a relationship; a discussion of the many factors affecting the serum PBI level is offered to support the contention that no such relationship should be anticipated. / Land and Food Systems, Faculty of / Graduate

Six male and six female chinchilla (Chinchilla lanigera) in the late phase of growth were used to study the effects of sex, crude protein level in the ration and duration of experiment on body weight gains, digestibility of energy, dry matter, organic matter and protein and efficiency of protein utilization, as measured by biological value and net protein utilization. Two isocaloric rations of differing crude protein content (16.25% and 19.56%) were supplied ad libitum for three one-week experimental periods. The results showed that female chinchilla had significantly (P<0.05) greater body weight gains than males after adjustment for initial body weight and feed intake. There was a significant (P<0.05) effect of ration on the digestibility coefficients studied. The mean apparent digestibility coefficients for energy, dry matter, organic matter and protein for ration 1 (16.25% crude protein) were 65.09, 66.44, 67.73 and 62.83%, respectively; while for ration 2 (19.56% crude protein) the values were 67.32, 68.52, 70.21 and 73.23%, respectively. On the other hand, sex had no significant (P<0.05) effect on digestibility. There was a significant (P<0.05) effect of ration on the protein utilization indices studied. Biological value was not significantly (P<0.05) different for the two rations. The mean values for biological value and net protein utilization for ration 1 (16.25% crude protein) were 66.38 and 42.02%, respectively; while for ration 2 (19.56% crude protein) the values were 66.96 and 48.17%, respectively. On the other hand, sex had no significant (P<0.05) effect on protein utilization. The sensitivity of growing chinchilla to protein quality suggests a major role for prececal digestion and absorption although this does not preclude the synthesis and subsequent breakdown of microbial protein in the postcecal part of the gut. / Land and Food Systems, Faculty of / Graduate

Rubella virus is a small enveloped positive strand RNA virus. Two species of viral RNA are found in infected cells: a full-length genomic RNA and a subgenomic species corresponding to the 3' one third of the genomic RNA molecule. The 24S subgenomic RNA specifies a polyprotein which is cotranslationally processed by endoproteolytic cleavage by host signal peptidase to yield three structural proteins, El, E2 and capsid. El and E2 are membrane glycoproteins forming the virion spikes, and C protein binds to 40S genomic RNA to form a nucleocapsid. El and E2 proteins contain N-linked oligosaccharide as a consequence of their passage through the endoplasmic reticulum (ER) and Golgi apparatus. According to the signal hypothesis, translocation of secretory and membrane proteins into the ER is mediated by a hydrophobic signal peptide. The signal peptides for E2 and El have been localized by in vivo expression of El and E2 cDNAs. Oligonucleotide-directed mutagenesis was used to define the cleavage sites between C, E2, and El, as well as the effect of the cleavages on the transport and processing of E2 and El. The expression of the cleavage site mutants was studied in vitro and in vivo. It was found that uncleaved precursor polypeptides were retained in the ER and very little E2 or El polypeptide was observed at either the Golgi apparatus or the plasma membrane. The E2 and El polypeptides can cross the ER membrane without the cleavage of the signal peptide while the transport of E2 and El beyond the ER requires the cleavage of E2 from C and El from E2. The C-termini of the C and E2 proteins, which were not previously defined, have been partially characterized. Capsid protein does not appear to undergo further proteolytic processing after it is cleaved from E2 by signal peptidase, but E2 may be processed at a second cleavage site at its C-terminus by a trypsin-like enzyme. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

No description available.

The metabolic response to muscle unloading and disuse was studied in rats subjected for six days to tail-cast suspension which leads to non-weight bearing hindlimbs, while the forelimbs are utilized for mobility. Under these conditions the soleus muscle atrophied, growth of the gastrocnemius and plantaris muscles declined, and the extensor digitorum longus and tibialis anterior muscles grew normally. Differences in muscle weight and protein content were associated with slower protein synthesis, particularly in the sarcoplasmic fraction, and faster protein degradation. Atrophy was accentuated by the administration of glucocorticoids to adrenalectomized rats causing a further reduction in protein synthesis. The effects of disuse and glucocorticoids were additive. Amino acid metabolism is altered in disuse of the soleus muscle as demonstrated by higher concentrations of tyrosine and glutamate and lower concentrations of glutamine, aspartate and asparagine. The lower glutamine concentration is the result of slower de novo synthesis despite higher glutamine synthetase activity, the result of glucocorticoid action. Slower glutamine synthesis was due to a lack of free ammonia for its synthesis, probably due to slower flux through AMP deaminase as a result of decreased muscle use and ATP utilization. Alanine production by the atrophied soleus muscles was higher and glutamate and aspartate utilization lower than by muscles from weight bearing controls. Branched chain amino acid degradation is faster in both the soleus and extensor digitorum longus muscles of suspended rats; a difference partially mediated by glucocorticoids. The resistance of amino acid uptake to insulin in these muscles was abolished in adrenalectomy. Passive stretch of the non-weight bearing soleus muscle, by immobilization in dorsiflexion, prevented the metabolic changes associated with hindlimb suspension while plantar flexion had no effect on the atrophy. Muscles subjected to hypokinesia/hypodynamia demonstrated a loss of muscle protein due to slower protein synthesis and faster degradation. The increased availability of free amino acids from protein coupled to differences in hormone responsiveness and slower utilization of energy stores results in a substantial shift in the metabolic equilibrium of the muscle cell, particularly with regard to amino acid interconversions.

by Kwok-keung Chan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 177-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Skeletal muscle proteins are the largest amino acid pool in animal body and are continuously degraded and resynthesized. Dozens of factors have been shown to influence the balance between synthesis and degradation and thereby regulate muscle growth and function. It is now know that one of the major regulatory bases of muscle metabolism is neuron-muscle interaction. A neurogenic factor, ciliary neurotrophic factor (CNTF), is proposed to exert myotrophic actions and could possible be a mediator of neuron-muscle interactions. The goal of this study was to investigate the effects of CNTF on muscle protein turnover in an in vitro system. CNTF was applied to differentiated cultured muscle cells (L8 cell line). Radiochemical labeled amino acids were added to the culture medium to determine the rate of incorporation or release by the cells as indexes of protein synthesis and protein degradation, respectively. Total protein was measured as an index of change in total protein accretion. Twelve hours of CNTF treatment increased myofibrillar protein synthesis by 10%. However, longer CNTF treatment (24 hours) reduced non-myofibrillar protein synthesis. CNTF (1 ng/ml) decreased protein degradation but higher doses (20 ng/ml) accelerated protein degradation. These changes in protein turnover resulted from changes in the myofibrillar protein fraction rather than from changes in turnover of the non-myofibrillar fraction. The change in protein synthesis and protein degradation resulted in an increase in total protein accretion of about 6%. Compared with the myotrophic studies on the effects of CNTF in vivo, the action of CNTF were relatively small. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that CNTF receptor alpha subunit (CNTFR��) mRNA expression is lower than which is expressed in muscle tissue. This could explain the reason why CNTF exerted smaller effects in vitro compared to those reported in vivo. Overall, CNTF exerts a small but statistically significant anabolic actions in cultured skeletal muscle and the actions were highly dose-dependent. The limited action of CNTF in vitro may be related to its low receptor density in the L8 cell (compared to in vivo). Because actions may be highly dose-dependent, a challenging series of studies are ahead for anyone wishing to identify the signal transduction mechanisms which account for CNTF's actions. / Graduation date: 1998