Intracellular protein breakdown in a thermophile

Protein breakdown of 5 to 7% per hr was found in nitrogen-starved cells of an unclassified prototrophic thermophilic bacillus; a similar protein-breakdown rate (6.5% per hr) was found in resting cells of Escherichia coli. In the thermophile, the rate of protein breakdown was markedly influenced by the temperature; it was maximal between 45 and 55 C, and it decreased considerably at 35 and 75 C, temperatures which are only slightly below or above the minimal and maximal growth temperatures. Growing cultures of the thermophile showed little, if any, protein breakdown, a finding similar to that of others with E. coli.     

Measurement of muscle protein fractional synthesis and breakdown rates from a pulse tracer injection

We have developed a new method to determine the fractional synthesis rate (FSR) and breakdown rate (FBR) of muscle protein. This method involves a pulse tracer injection and measurement of enrichment in the arterial blood and muscle at three time points. The calculations of FSR and FBR are based on the precursor-product principle. To test this method, we gave a pulse injection of L-[ring-(13)C(6)]phenylalanine of 4-6 mg/kg in five rabbits. The measured FBR value (0.233 +/- 0.060%/h) was almost identical (P = 0.35) to that (0.217 +/- 0.078%/h) estimated from a leg arteriovenous balance model (Biolo G, Chinkes D, Zhang X-J, and Wolfe RR. J Parenter Enteral Nutr 16: 305-315, 1992). The measured FSR value tended to be lower than that estimated from the leg model (0.125 +/- 0.036 vs. 0.185 +/- 0.086%/h; P = 0.14), possibly because the new method measures only muscle FSR, whereas the leg balance model also includes skin and bone contributions. The pulse tracer injection did not affect muscle protein kinetics as measured by leucine and phenylalanine kinetics in the leg. In another five rabbits, we demonstrated that sampling could be reduced to either one or two muscle biopsies when multiple pulse injections were used. This method can be completed in 1 h with one muscle biopsy and has technical advantages over currently used methods.     

Measurement of protein turnover in rat brain

Abstract— Degredation rates of rat brain proteins were measured by following the decay in specific radioactivity of carboxyl labelled aspartate and glutamate over a 17-day period. Initial labelling of these amino acids was achieved by a single intraperitoneal injection 0f NaH¹⁴CO₃. The non-linear decay curve for total brain proteins could be approximated by assuming that the mixture contained two classes of proteins with half-lives of 3.3 and 8.7 days, respectively. Half-lives of 2.5 and 7.7 days were estimated for such protein classes in the microsomal fraction. The half-lives of soluble proteins, synaptic membranes, cell body and synaptic mitochondria were 3.1, 5.8, 5.6 and 8.4 days, respectively. Identical results were obtained if the change in specific activity of intact protein labeled by NaH¹⁴CO₃ was followed. Two-fold slower decay rates were obtained when brain proteins were labeled with a pulse of [4,5-³H]leucine or [l-¹⁴C]leucine. Half-lives calculated for the two classes of proteins in whole brain were 8.4 and 16.5 days, respectively with [4,5-³H]leucine and 8.9 and 14.2 days, respectively with [1-¹⁴C]leucine. These results indicate the very significant reutilization of this amino acid in brain. Sodium [¹⁴C]bicarbonate is a more satisfactory isotopic precursor for accurate assessment of rates of protein turnover in brain.     

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle

N^t-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with $\text{[}{}^{\mathrm{Me}}\text{-}{}^3\text{H}\text{]}\text{methionine}$. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.     

Comparison of three different supercharging procedures in a rat skin flap model

A significant clinical problem in reconstructive surgery is partial loss of a pedicled flap. To resolve this problem, various methods of vascular augmentation have been developed; "supercharging" is one of those techniques. A new rat flap model was developed for investigation of the supercharging procedure, and the efficacy of the arterial supercharging method was examined. The purpose of this study was to investigate how an arterial supercharging procedure could generate large flap survival areas with different supercharging positions in rats. On the basis of the vascular anatomical features of rats, a circumferential skin flap from the lower abdomen to the back, measuring 4 x 12 cm, was marked. The flap was divided along the dorsal midline. Forty rats were divided into four experimental groups, as follows: group 1 (control), flaps based only on the deep circumflex iliac artery and vein; group 2, flaps supercharged with the ipsilateral superficial inferior epigastric artery; group 3, flaps supercharged with the contralateral superficial inferior epigastric artery; group 4, flaps supercharged with the contralateral deep circumflex iliac artery. On the fourth postoperative day, the flaps were evaluated with measurements of necrosis and survival areas. Microfil (Flow Tech, Inc., Carver, Mass.) was then injected manually throughout the body, and the vascular changes produced by supercharging were angiographically evaluated. Compared with group 1 (control), the flap survival areas were significantly greater in distally supercharged flaps in groups 3 and 4 (mean flap survival, 91.2 +/- 5.2 percent and 90.5 +/- 10.6 percent, respectively; p < 0.001) and in proximally supercharged flaps in group 2 (45.9 +/- 4.1 percent, p < 0.05). Angiographic assessment of the flaps that survived completely revealed marked dilation of the choke veins among the territories and reorientation of dilated veins along the axes of the flaps. This study suggests that distal arterial supercharging (contralateral superficial inferior epigastric artery or contralateral deep circumflex iliac artery) is more effective than proximal arterial supercharging (ipsilateral superficial inferior epigastric artery) in increasing flap survival. Although the rat skin flap may not be analogous to human flaps, distal arterial supercharging might have useful therapeutic potential in increasing flap survival in clinical practice.     

Effects of corticosterone on connectin content and protein breakdown in rat skeletal muscle

We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Dietary protein as a potent regulator of the hyaluronan synthase gene in rat skin

The status of hyaluronan, the major glycosaminoglycan in the skin, is regulated by many factors such as cytokines and glucocorticoids. To examine whether and how protein malnutrition affects the status of skin hyaluronan, the hyaluronan content and mRNA levels of hyaluronan synthases (has) were analyzed in the skin of rats fed on a protein-free diet or on a 12% gluten diet. When these malnourishing diets had been given for 1 week, the hyaluronan content was significantly reduced as compared with that in rats fed on a 12% casein diet. Substantial falls in the mRNA levels of rhas2 and rhas3 were also observed. The reduction of mRNAs was already evident on the second day of treatment with the malnourishing diets. These results suggest that protein malnutrition has a primary impact on the gene expression of rhass, which leads to the reduction of hyaluronan content and to disfunction of the skin.     

Exploring the mechanisms of in vivo regulation of liver protein breakdown in the rat by the use of the new antilipolytic-agent model.

In this study, we explored the changes in the rate of protein degradation in liver cells in vivo, using a method based on the physiological stimulation of liver autophagy. Male albino rats 1, 2, 6, 12 and 24 months old were fasted overnight, and then received an injection of the antilipolytic agent 3,5-dimethylpyrazole (DMP) to evoke a sudden shortage of lipid fuel. A comparison was made with the in vivo effects of glucagon by giving the 2-month-old group an intraperitoneal injection of this hormone. Samples of liver were taken after 0, 15, 20, 30, 60 and 150 min and processed for electron microscopy, and groups of rats were subjected to short-term single pass liver perfusion. Results show that in the younger age-groups, the DMP stimulation of liver autophagy and amino acid release is highly significant, and compares favourably with the glucagon model of induction of the autophagic process. With older rats, an age-related longer time-lag of the autophagic response and a decrease in the effect of DMP were observed. In conclusion, hormones may activate autophagy, whereas levels of plasma amino acids may tune down the process to adjust the availability of the substrate to tissue needs.     

Measurement of a folate binding protein from rat liver cytosol by radioimmunoassay

A radioimmunoassay has been developed for the folate binding protein from rat liver cytosol with a molecular weight of 150,000 which was recently purified to homogeneity (Suzuki, N., and Wagner, C., 1980, Arch. Biochem. Biophys.199, 236–248). This method has indicated that the binding protein (FBP-CII) is found primarily in the liver. A significant amount of FBP-CII was also found in the kidney and much reduced levels in spleen, serum, brain, lung, and heart. No FBP-CII could be detected in small intestine, skeletal muscle, or testes. Small amounts of cross-reacting material were found in the livers of mouse, dog, chick, and humans. Levels of FBP-CII were not decreased in the livers of folate-deficient rats. Assays of rat fetal liver and kidney 2 days prior to birth showed much lower levels which increased rapidly at birth. These data are consistent with the FBP-CII fulfilling a role as a folate storage protein in rat liver.     

Aging changes in intracellular protein breakdown

Using radioactively labelled cytosol proteins as substrates we were able to exclude the possible accumulation of any specific inhibitor for the lysosomal proteases in rat liver cytosol during the aging process. There were also no gross changes in the molecular weight patterns of these proteins during the aging process. The percentage of more hydrophobic proteins seems to be identical in both the "old" and "young" cytosol proteins. From immunological experiments we suppose a qualitative change in the composition of rat liver cytosol proteins or of their properties during the aging process.     

Angiogenesis and growth factor modulation induced by alternagin C, a snake venom disintegrin-like, cysteine-rich protein on a rat skin wound model

In this work, we show that alternagin-C (ALT-C) and ALT-C PEP, a peptide derived from its sequence, were able to induce angiogenesis in wounded rat skin. A spherical cutaneous excision was made in the back of each animal and treated with three different concentrations of ALT-C or ALT-C PEP. After that, the skin was removed and analyzed to verify the presence of new vessels and the expression of growth factors. ALT-C and ALT-C PEP induced the formation of new vessels and modulated the expression of growth factors, mainly VEGF and FGF1. The expression of VEGF increased and it could be detected up to 7 days after injury. FGF1 also significantly increased, but at a lesser extent than VEGF. In conclusion, the present study shows for the first time the stimulation of angiogenesis in an injured tissue by a disintegrin-like protein and that ALT-C may exert this effect by modulating the expression of growth factors.     

Effect of a high protein diet on glucose tolerance in the rat model

The purpose of this study was to determine the effects of a high protein diet on glucose tolerance. Nine Sprague Dawley rats received a high protein (HP) diet (65% protein, 35% fat) and eight rats consumed a standard chow (SC) diet over eight weeks. Oral glucose tolerance tests (OGTT) were performed at the end of the third and the seventh week. The diet did not effect glucose tolerance in the first (SC=10357+/-294 mg/dl/120 min; HP=9846+/-300 mg/dl/120 min) or the second OGTT (SC=10134+/-395 mg/dl/120 min; HP=10721+/-438 mg/dl/120 min) as reflected by the area under the glucose concentration curve. Similarly, the area under the insulin concentration curve was not effected by the high protein diet during the first (SC=49.21+/-8.46 ng/ml/120 min; HP=41.75+/-10.54 ng/ml/120 min) or the second OGTT (SC=96.63+/-13.68 ng/ml/120 min; HP=92.77+/-17.44 ng/ml/120 min). The high protein diet group experienced a delayed glucose response for the first (SC=30 min at 112+/-7 mg/dl; HP=60 min at 101+/-5 mg/dl) and second OGTT (SC=15 min at 117+/-5 mg/dl; HP=60 min at 95+/-7 mg/dl). Body mass increased to the same extent in each diet group from the initial to final weighing (SC=159+/-2 g to 254+/-7 g; HP=157+/-2 g to 242+/-7 g). Despite a delay in peak glucose response, these findings suggest that glucose tolerance and body mass were neither adversely nor positively affected by a high protein diet.     

Plasma-waveguide model of electric breakdown in gas

Drawbacks of the conventional model of electric breakdown in high-pressure gases are discussed. A new model that associates the propagation of a breakdown wave with the propagation of a traveling electromagnetic wave in a plasma waveguide is proposed. Based on the new model, the main physical parameters of a medium are estimated.     

Pepstatin- and leupeptin-loaded liposomes: a tool in protein breakdown studies

The preparation of proteinase inhibitor loaded "solid" positively charged liposomes is described. Some properties of the vesicle preparations are given and the findings are tentatively summarized in a scheme of liposomophagy.