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Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells. This mRNA was translated in the proliferating myoblasts but not in the non-proliferating myotubes. A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle-specific genes in mitogen-poor differentiation medium. In the absence of any detectable accumulation of muscle-specific α-actin mRNA, the P40 mRNA remained in the translated state. A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle-specific actin and tropomyosin mRNAs. On the other hand, it showed no effect on the translation of P40 mRNA. Addition of Ca2+ to the EGTA-treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days. Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non-translated state. An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle-specific mRNAs. The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA-dependent micrococcal-nuclease-treated homologous cell-free extracts. The extracts from myoblasts and myotubes were able to translate P40 mRNA. Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs. Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA. The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing these ribosomes with a high-ionic-strength buffer. In order to elucidate the role of interaction between mRNA and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA-protein (mRNP) complexes were also examined. Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of the RNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells. Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein than polysomal P40 mRNP. Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNP complexes showed that they were composed of identical polypeptides. The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP. [ABSTRACT FROM AUTHOR]

The mRNA coding for a 40-kDa polypeptide (P-40) was previously cloned and sub-cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59-67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non-dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non-polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600-base-pair Pst1 fragment of the recombinant clone was 3'-end-labeled with cordycepin 5'-[α32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600-nucleotide-long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3' ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P-40 mRNA. The result of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P-40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P-40 mRNA in myotubes. The relationship between the translation of P-40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady-state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of ³H-labeled P-40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P-40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P-40 mRNA was synthesized in both myoblasts and myotubes. Furthermore, the α-amanitin sensitivity of the synthesis of P-40 mRNA was examined using a nuclear transcription system. The result of this analysis has shown that synthesis of P-40 mRNA was sensitive to a low concentration of α-amanitin, suggesting that P-40 mRNA, like other mRNAs, is also transcribed by RNA polymerase II. [ABSTRACT FROM AUTHOR]

In muscle cells two populations of mRNA are present in the cytoplasm. The majority of mRNA is associated with ribosomes and active in protein synthesis. A small population of cytoplasmic mRNA occur as free mRNA- protein complex and is not associated with ribosomes. This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library. Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA. One of these clones was extensively studied. The DNA from a recombinant plasmid D12 hybridized to a 1.3-kb poly(A)-rich mRNA. In proliferating myoblast cells, the 1.3-kb mRNA was more abundant in the polysomal fraction and mostly free in the non-dividing myotubes. In contrast to this mRNA. 90% of α and β actin mRNAs were translated in both myoblasts and myotubes. Further analysis of distribution of the 1 .3-kb RNA in the polysomal (active) and free (repressed) fractions in fusion-arrested post- mitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA. Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA. The presence of this mRNA was not, however, limited to muscle cells. This mRNA was also present in the repressed state in rat liver and kidney cells. These results, therefore, suggest that the 1.3-kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non-dividing cells. Messenger RNA homologous to the 600-base-pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes. Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell-free system synthesized a 40-kDa polypeptide. These results suggest that the repressed population of 1.3-kb mRNA can be translated in vitro. The hybridization pattern of DNA from the recombinant plasmid 1)12 with rat genornic DNA suggested that the 1.3-kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome. [ABSTRACT FROM AUTHOR]

Examines the distribution of messenger RNA (mRNA) between the detergent-soluble and insoluble (cytoskeleton) fractions in rat L6 myoblast and myotube cells. Association of cytoplasmic in both myoblasts and myotubes with the cytoskeletal framework; Presence of cytoskeleton-bound mRNA polysomes; Nature of binding between mRNA and the cytoskeletal framework following in vivo crosslinking of RNA and protein by irradiating muscle cells with ultraviolet light.

Abstract: An one-pot reaction of Re2(CO)10 and an α-diimine (1:2 molar ratio) in refluxed 2-chloroethanol afforded the corresponding chlorotricarbonyl(α-diimine)rhenium(I) complexes, fac-(CO)3(α-diimine)ReCl, 1-4, in very high yield. The α-diimines in 1-4 are 2,2′-bipyridyl, 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline, and 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, respectively. Alternatively, an one-pot reaction of Re2(CO)10 and an α-diimine (1:2 mole ratio) in refluxed 1-pentanol in the presence of CO2 and HCl yielded the corresponding chloro complexes, 1-4, in very high yield. 1-4 were characterized spectroscopically. Additionally, 3 and 4 were characterized through X-ray crystal structure determinations. The chloro complexes, 1-4 exhibit fluorescence both in the solid states and in solutions. Surprisingly, 1-4 are cytotoxic against breast, prostate, and lung cancer cell lines. [Copyright &y& Elsevier]

Purpose: Oxalate, a metabolic end product, is a major constituent of majority of renal stones. Previous studies with LLC-PK1 cells, a line of proximal renal epithelial cells of porcine origin, have shown that oxalate produces time and concentration dependent effects on the growth and viability of these cells. We assessed the possibility that oxalate may be toxic to HK-2 cells, a line of human proximal renal epithelial cells., Materials and Methods: HK-2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and antibiotics. Cells were exposed to oxalate for various intervals. Trypan blue exclusion criteria were used to assess membrane integrity, cell morphology was assessed by hematoxylin and eosin staining and crystal violet staining was used to measure cell density. DNA synthesis was measured by [3H]-thymidine incorporation and superoxide production was measured by the nitroblue tetrazolium reduction method., Results: Exposure of HK-2 cells to oxalate produced time and concentration dependent increase in the membrane permeability to trypan blue and changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in an increase in DNA synthesis and alterations in cell viability with net cell loss after exposure to high oxalate concentrations., Conclusions: To our knowledge the results provide the first direct demonstration of the toxic effects of oxalate in HK-2 cells, a line of human renal epithelial cells, and suggest that hyperoxaluria may contribute to renal tubular damage associated with calcium oxalate stone disease.