The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca 2؉ -binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca 2؉ ). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca 2؉ stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca 2؉ -free (apo) CaM without ubiquitination. Addition of Ca 2؉ dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca 2؉ binding, native CaM assumes a non-degradable conformation, which most of the agedamaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

Panax quinquefolium L. (American Ginseng, AG) is an herb characteristic for regions of North America and Asia. Due to its beneficial properties it has been extensively investigated for decades. Nowadays, it is one of the most commonly applied medical herbs worldwide. Active compounds of AG are ginsenosides, saponins of the glycosides group that are abundant in roots, leaves, stem, and fruits of the plant. Ginsenosides are suggested to be primarily responsible for health-beneficial effects of AG. AG acts on the nervous system; it was reported to improve the cognitive function in a mouse model of Alzheimer’s disease, display anxiolytic activity, and neuroprotective effects against neuronal damage resulting from ischemic stroke in animals, demonstrate anxiolytic activity, and induce neuroprotective effects against neuronal damage in ischemic stroke in animals. Administration of AG leads to inhibition of hypertrophy in heart failure by regulation of reactive oxygen species (ROS) in mice as well as depletion of cardiac contractile function in rats. It also has an anti-diabetic and anti-obesity potential as it increases insulin sensitivity and inhibits formation of adipose tissue. AG displays anti-cancer effect by induction of apoptosis of cancer cells and reducing local inflammation. It exerts antimicrobial effects against several pathogenic strains of bacteria. Therefore, AG presents a high potential to induce beneficial health effects in humans and should be further explored to formulate precise nutritional recommendations, as well as to assess its value in prevention and therapy of some disorders, including cancer.

Functional comparison of isolated hearts with different sizes has been difficult because function varies at different ventricular volumes. To date, no standard volume has been established. To determine the most accurate experimental approach, we tested five different methods to standardize volume in control hearts with different sizes but similar papillary muscle function and in hearts with concentric hypertrophy: intracardiac balloon volume (VB) = 120 microliters (M1). VB at diastolic pressure = 10 mmHg (M2) or diastolic wall stress = 4 kdyn/cm2 (M3), V1 = 25 microliters/100 g body weight (M4) and VB = 50% of volume at peak developed pressure (Vmax; M5). Systolic and diastolic functions of control groups were different using M1 and comparable using M2 or M5. M3 and M4 showed borderline significant differences. We concluded that M5 and M2 were suitable to compare function among hearts of different sizes. If diastolic compliance is of interest, as in concentric hypertrophy, parameter-volume curves should be normalized by Vmax to compare function at corresponding points of the Frank-Starling curve (e.g., at 50% of Vmax, M5).

One of the most effective strategies to enhance metabolite biosynthesis and accumulation in biotechnological systems is the use of elicitation processes. This study assesses the influence of different concentrations of yeast extract (YE) on ginsenoside biosynthesis in Panax quinquefolium (American ginseng) hairy roots cultivated in shake flasks and in a nutrient sprinkle bioreactor after 3 and 7 days of elicitation. The saponin content was determined using HPLC. The maximum yield (20 mg g−1 d.w.) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved after application of YE at 50 mg L−1 concentration and 3 day exposure time. The ginsenoside level was 1.57 times higher than that attained in control medium. The same conditions of elicitation (3 day time of exposure and 50 mg L−1 of YE) also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The total ginsenoside content was 32.25 mg g−1 d.w. and was higher than that achieved in control medium and in shake flasks cultures. Obtained results indicated that yeast extract can be used to increase ginsenoside production in hairy root cultures of P. quinquefolium.

To determine whether densities of calmodulin (CaM) and CaM-binding proteins are related to phasic and tonic behavior of smooth muscles, we quantified these proteins in the opossum esophageal body (EB) and lower esophageal sphincter (LES), which represent phasic and tonic smooth muscles, respectively. Gel electrophoresis, immunoprecipitation, Western blot, and hemagglutinin epitope-tagged CaM (HA-CaM) overlay assay with quantitative scanning densitometry and phosphorylation measurements were used. Total protein content in the two smooth muscles was similar (∼30 mg protein/g frozen tissue). Total tissue concentration of CaM was significantly (25%) higher in EB than in LES ( P < 0.05). HA-CaM-binding proteins were qualitatively similar in LES and EB extracts. Myosin, myristoylated alanine-rich C kinase substrate protein, Ca2+/CaM kinase II, and calponin contents were also similar in the two muscles. However, content and total activity of myosin light chain kinase (MLCK) and content of caldesmon (CaD) were three- to fourfold higher in EB than in LES. Increased CaM and MLCK content may allow for a wide range of contractile force varying from complete relaxation in the basal state to a large-amplitude, high-velocity contraction in EB phasic muscle. Increased content of CaD, which provides a braking mechanism on contraction, may further contribute to the phasic contractile behavior. In contrast, low CaM, MLCK, and CaD content may be responsible for a small range of contractile force seen in tonic muscle of LES.