José Hernández-Yago

Use of rhodamine 123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria

The fluorescent dye Rhodamine-123, which selectively stains mitochondria depending on the mitochondrial membrane potential, was used with flow cytometry to evaluate alterations in activity of mitochondria isolated from mouse liver. Under in vitro conditions, with succinate and ADP present in the buffer, mitochondrial activity was affected by a variety of metabolic inhibitors that modify membrane potential. These results demonstrate clearly that flow cytometric techniques using Rhodamine-123 can be employed to study activity in isolated mitochondria.

Regulation of lysosomal autophagy in transformed and non-transformed mouse fibroblasts under several growth conditions

The role of the lysosomal system in accelerated protein degradation was investigated in 3T3 mouse fibroblasts and in the SV40 virus-transformed derivative, SV3T3. Rates of protein degradation and quantitative electron microscopic alterations in the lysosomal system were compared under four different growing conditions: exponential growth, confluent phase, serum deprivation, and confluent phase together with serum deprivation. We found a significant correlation between increases in rates of proteolysis of long-lived proteins and fractional volume of lysosomes, suggesting a causal relationship between the two, as well as a morphological explanation for the differences in rates of protein degradation in transformed and non-transformed cultured cells. The increase in lysosomal fractional volume resulted from an increase in dense bodies only (in serum-deprived exponential or confluent cultures) or from an increase in autophagic vacuoles and dense bodies (in serum-supplemented confluent cultures).

Significance of the rapid degradation of newly synthesized proteins in mammalian cells: A working hypothesis☆

Abstract The decay curve for labelled proteins in living tissue (biomass) is the integral of the different slopes for every species of protein present. While this heterogeneity may account for some of the complexity of degradation kinetics, a factor which must play an important role—especially where discontinuities in exponential decay curves occur—is the stabilization of newly synthesized (nascent) proteins. These metastable proteins remain at risk and experience a high rate of proteolysis until they become integrated into the biomass. Following the initial period of stabilization, integrated proteins randomly but steadily emerge under controlled conditions, reflecting their individual turnover rate in the biomass. Destabilized proteins will be susceptible once again to proteolysis, but the rate of their degradation will be slower than in the initial phase because the frequency of their re-emergence from their sites of integration has become the rate limiting factor. Irreversible conformational changes and denaturation may further influence their rate of removal. Recognition of the two distinct phases of proteolysis offers a better explanation of the kinetics of degradation of endogenous proteins. Several important implications arising out of these findings are discussed.

Rats fed prolonged high protein diets show an increase in nitrogen metabolism and liver megamitochondria

Rats were fed diets containing 20, 50 and 80% protein for 14 months. The urea excreted by the rats fed diets containing 50 and 80% protein when compared to rats fed diets containing 20% protein increased ca. 2- and 3-fold, respectively, in ca. 2 days; this increase was maintained essentially unchanged through the experimental period. The serum levels of urea increased 2.5- and 4-fold, respectively, in the first days and were also maintained during the experiment. Glutamate dehydrogenase activity of liver remained unchanged. The five urea cycle enzymes increased with respect to the control values. Orotic acid excretion increased as well as orotidylate decarboxylase and orotate phosphoribosyltransferase, but aspartate transcarbamylase did not. The key amino acids involved in the urea and pyrimidine pathways in liver were also measured; aspartic and glutamic acids and citrulline were increased, and ornithine and arginine did not change with the higher protein intake. In general, no differences were observed between animals fed 50 and 80% protein in their diets. Protein synthesis did not increase with the increase of protein content of the diet. Stereological analysis of ultrathin sections showed that the high protein diet induced a significant increment in the volumetric density, numerical density and size of hepatocyte mitochondria. Moreover, the presence of giant mitochondria, a hundred times larger than normal, was also observed in some periportal hepatocytes of rats fed the 80% protein diet.

Role of Thyroid Hormone in Craniofacial and Eye Development Using a Rat Model

Thyroid hormones (TH) are essential for somatic and neural development. Epidemiological studies have pointed to TH-dependent craniofacial features occurring during development. In an attempt to elucidate the precise role of TH in the developing eyes and adnexa (orbit, lids, nasolacrimal structures), we analysed the craniofacial and eyeball developmental characteristics in a rat model of congenital-neonatal hypothyroidism (HG), induced by combined chemical-surgical thyroidectomy. The heads and eyeballs from control and HG animals were obtained at key developmental stages and processed for scanning electron, light and transmission electron microscopy. On embryological day 13 (E13), significantly reduced values for head parameters (25% less), optic primordia area (0.053 ± 0.0085 vs. 0.111 ± 0.012 µm2; p < 0.05) and volume (3.96 ± 0.141 vs. 8.09 ± 0.123 µm3; p < 0.05) were found in the HG with respect to the controls. In addition, a delayed prenatal eye closure and postnatal eye opening took place in the treated rats. The photoreceptor and ganglion cell layer thickness displayed significantly lower values (p < 0.001) in HG, at each developmental time point. Postnatally, a delay in photoreceptor outer segment morphogenesis (in relation to retarded disc formation) and significantly lower values for ganglion cell nuclear volumes (p < 0.001) and nuclear pore density (p < 0.01) were observed in the TH-deficient animals. All data suggest that TH play a pivotal role in the development of the face and eye. Therefore, a series of defects due to a loss of TH actions involved in anterior-posterior development of the head and face and the loss of TH-dependent signals crucial for cell differentiation, migration, proliferation and establishment of definitive cell phenotypes in the eyes may appear. Gestational and neonatal screenings for thyroid functioning are suggested to paediatricians and ophthalmologists in order to prevent craniofacial malformations and visual abnormalities.

Regulation of mitochondrial protein concentration: a plausible model which may permit assessing protein turnover.

Publisher Summary This chapter focuses on regulatory controls of mitochondrial protein turnover and measurement of mitochondrial protein degradation. Cell metabolism is mediated by the activity of many enzymes and is controlled by the regulation of key enzyme activities and enzyme levels. Most mitochondrial proteins are coded by nuclear genes and synthesized on cytosolic ribosomes as larger precursors, which then migrate and are imported into mitochondria. The overall protein concentration per cell volume changes very little, although the concentration of individual proteins may do so temporarily. Therefore, protein synthesis and degradation should be closely interrelated. The in vitro protein concentration in mitochondria may be regulated by the entry of mitochondrial protein precursors from the cytosol. Enzymes are flexible molecules whose conformations can be altered upon binding of substrates or other ligands. Mitochondrial proteins are degraded by lysosomes because mitochondria are frequently seen inside autophagic vacuoles. The importance of protein degradation is chiefly as a process to control the concentration of a protein and, thus, cell metabolism. Protein synthesis is usually controlled by regulating the amount of its messenger RNA available for translation.

Fate of proteins synthesized in mitochondria of cultured mammalian cells revealed by electron microscope radioautography

Abstract Synthesis of mitochondrial proteins was studied in a monkey kidney epithelium cell line by electron microscope radioautography after pulse label for 1 h with [3H]leucine in the presence of cycloheximide followed by chase in the absence of the antibiotic. At all chase times tested the highest specific activities were found over mitochondria and lysosomes (dense bodies). Based on regression analysis of the decay curve of mitochondrial labeling, there appeared to be two main mitochondrial populations with apparent half-lives of 1–2 h and 6 days. In parallel experiments, cells were labelled with [3H]leucine in the presence of cycloheximide plus chloramphenicol. Chloramphenicol markedly inhibited labelling over mitochondria and dense bodies, whereas it had no significant effect on labelling of other cell compartments (nucleus, endoplasmic reticulum, remainder cytoplasm). The significance of the cycloheximide-insensitive and chloramphenicol-sensitive lysosomal labelling is discussed. The data suggest that in cultured cells lysosomes are involved in the degrading of mitochondrial proteins.

Cell-free synthesis and processing of a large precursor of glutamate dehydrogenase of rat liver

Abstract The mitochondrial matrix protein glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using mRNA from free or membrane-bound polysomes from rat liver. Immunoprecipitation of the ( 35 S)methionine labeled translation mixture was performed using rabbit anti-glutamate dehydrogenase serum. Analysis after electrophoresis of the immunoprecipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed that the glutamate dehydrogenase is synthesized ‘in vitro’ as a large precursor. A mitochondrial extract from rat liver processed the precursor synthesized “in vitro” to the mature form.

Spermidine and spermine stimulate the transport of the precursor of ornithine carbamoyltransferase into rat liver mitochondria

We have examined the effect of low molecular weight components of the transport mixture generally used for the import of rat liver pre-ornithine carbamoyltransferase by isolated rat liver mitochondria. These studies revealed that spermidine and spermine, at physiological concentrations, stimulate the transport of the precursor of ornithine carbamoyltransferase into mitochondria. This stimulatory effect of spermidine and spermine is concentration-dependent and is completely inhibited at higher than physiological concentrations (20 mM for spermidine and 4 mM for spermine). Magnesium ions, which also have a stimulatory effect, inhibit the stimulatory effect of spermidine.

Exit of proteins and fragments thereof from mitochondria is accelerated by the import of cytosolic synthesized proteins

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria. Incubation of 35S-methionine labeled mitochondria from rat hepatocytes with proteins synthesized in a cell-free system, using messenger RNA from rat liver, dramatically increased the release of mitochondrial proteins and fragments thereof into the medium. Since the synthesized proteins include cytosolic precursors of mitochondrial proteins, our results strongly suggest that import of proteins from the cytosol into mitochondria influences the half-life of proteins in these organelles. The use of this simple approach--i.e. combining the study of protein import and exit with mitochondria--to further clarify intracellular protein turnover and its regulation is suggested.