Assessment Of Protein Synthesis Research Articles

Assessment of Protein Synthesis in Mitochondria Isolated from Rosette Leaves and Liquid Culture Seedlings of Arabidopsis.

Mitochondria are subcellular organelles with their own genome and expression system, including translation machinery to make proteins. Several independent studies have shown that translation is an essential regulatory step in expression of the plant mitochondrial genome. Thus, the study of mitochondrial translation seems to be crucial for the comprehension of plant mitochondrial biogenesis and maintenance. In organello protein synthesis in isolated mitochondria is a direct method to visualize the translational products of this organellar genetic system. In this method, highly purified, functional mitochondria synthesize proteins in the presence of radiolabeled amino acids, such as methionine, and an energy regeneration system. The labeled, newly synthesized polypeptides are separated by SDS-polyacrylamide gel electrophoresis and are detected by autoradiography. Here we describe the detailed protocol for in organello labeling of translation products that was optimized for mitochondria isolated from rosette leaves and liquid culture seedlings of Arabidopsis thaliana plants.

Stress-deprivation induces an up-regulation of versican and connexin-43 mRNA and protein synthesis and increased ADAMTS-1 production in tendon cells in situ

ABSTRACT Purpose: The proper function of the tenocyte network depends on cell-matrix as well as intercellular communication that is mechanosensitive. Building on the concept that the etiopathogenic stimulus for tendon degeneration is the catabolic response of tendon cells to mechanobiologic under-stimulation, we studied the pericellular matrix rich in versican and its predominant proteolytic enzyme ADAMTS-1, as well as Connexin-43 (Cx43), a major gap junction forming protein in tendons, in stress-deprived rat tail tendon fascicles (RTTfs). Materials and Methods: RTTfs were stress-deprived for up to 7 days under tissue culture conditions. RT-qPCR was used to measure mRNA expression of versican, ADAMTS-1, and Cx43. Protein synthesis was determined using Western blotting and immunohistochemistry. Results: Stress-deprivation (SD) caused a statistically significant up-regulation of versican, ADAMTS-1, and Cx43 mRNA expression that was persistent over the 7-day test period. Western blot analysis and immunohistochemical assessment of protein synthesis revealed a marked increase of the respective proteins with SD. Inhibition of proteolytic enzyme activity with ilomastat prevented the increased versican degradation and Cx43 synthesis in 3 days stress-deprived tendons when compared with non-treated, stress-deprived tendons. Conclusion: In the absence of mechanobiological signaling the immediate pericellular matrix is modulated as tendon cells up-regulate their production of ADAMTS-1, and versican with subsequent proteoglycan degradation potentially leading to cell signaling cues increasing Cx43 gap junctional protein. The results also provide further support for the hypothesis that the cellular changes associated with tendinopathy are a result of decreased mechanobiological signaling and a loss of homeostatic cytoskeletal tension.

Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains.

Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.

Assessment of protein synthesis and cellular proliferation in long‐lived crowded litter mice

Increasing the number of pups prior to weaning (“crowded litter” or CL) increases the lifespan of mice. In previous studies with caloric restriction, rapamycin feeding, and Snell dwarf mice, we have described the general phenomenon that DNA synthesis is decreased and mitochondrial (mito) protein synthesis is maintained, though this varies by tissue and model. Therefore, we hypothesized that CL mice would also have decreased DNA synthesis and maintained mito protein synthesis. UM‐HET3 mice were housed as litters of 8 (CON) or 12 (CL) prior to weaning. At 20 days of age pups were weaned onto a normal chow diet. At 4 months of age 4 CON and 4 CL animals had their drinking water supplemented with 8% deuterium oxide for 2 weeks to determine fraction of new total, cytosolic, and mito proteins, and fraction of new DNA. Protein synthesis was not statistically different in any tissue or fraction assessed. DNA synthesis was significantly greater in CL skeletal muscle (Con = 0.024±0.003, CL = 0.037±0.004), tended to be greater in heart (p=0.08; Con = 0.047±0.003, CL = 0.066±0.018), but did not differ in liver. CL mice were studied at a younger age than our previous investigations, and thus we conclude that either: 1) long‐lived models have a previously unrecognized increase in proliferation at young ages, or 2) CL mice are long‐lived through different mechanisms than our previously studied models.

Antimitotic and Cytotoxic Effects of Theophylline in MDA-MB-231 Human Breast Cancer Cells

A variety of cancer cell lines, including MDA-MB-231 human breast cancer cells, exhibit mitotic inhibition by cAMP. In earlier work, we found that the phosphodiesterase inhibitor, theophylline, reduced the number of cells and altered cellular morphology. In the current study, we evaluated the effects of theophylline on macromolecule synthesis and indices of cell viability. Theophylline evoked a concentration- and time-dependent decrease in DNA synthesis. However, the net decrease in cell number was greater than that predicted solely from mitotic arrest. Assessment of protein synthesis indicated a second effect of theophylline separable from that on DNA synthesis. This was confirmed by decreased cell viability and adhesion. Exposure of the cells to the phosphodiesterase inhibitor, IBMX, in concentrations that produced inhibition of DNA synthesis equivalent to that seen with theophylline, elicited a smaller reduction in cell number. Theophylline also evoked specific changes in the expression or function of membrane-bound adenylyl cyclase activity, effects that are likely to contribute to sustained reactivity of these cells to other cAMP-related inhibitors of cell proliferation, such as isoproterenol. The multiple pharmacologic properties of theophylline, producing mitotic inhibition, cytotoxicity and altered signaling in MDA-MB-231 cells, may provide insight into novel therapeutic strategies.

Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo.

In vivo microperifusion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo. Seminiferous and caput and cauda epididymal tubules were perifused for 3 h. with [35S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed. Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules. Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and pathophysiological effects on this important epithelial function in vivo.

Trichomonas vaginalis Resistance to Mengo Virus Infection*

We have investigated the susceptibility of Trichomonas vaginalis to Mengo virus infection by comparing the outcome of Mengo virus or purified Mengo virus RNA infection in T. vaginalis and in CCL-1 mouse fibroblasts. While the adsorption and entry of Mengo virus into T. vaginalis occurred in the same manner as in fibroblasts, the uncoating was much slower. In addition, Mengo virus infection of T. vaginalis displayed no eclipse nor any subsequent production of infectious virus. Purified RNA failed to initiate productive infection in T. vaginalis, whereas it provoked viral replication in the fibroblast controls. It was shown by assessment of protein synthesis in T. vaginalis and mouse fibroblasts cell-free systems that the protozoan ribosomes were able to translate endogenous mRNA and poly-U, but not viral RNA.