Linfadenectomía lumboaórtica primaria abierta v/s laparoscópica en pacientes con tumor testicular no seminomatoso, etapa I / Open primary lumboaortic lymphadenectomy v/s laparoscopic in patients with non (seminomatoso) testicular tumor: step 1

Análisis de la serie histórica de linfadenectomía lumboaórtica abierta comparándola con la serie de linfadenectomía laparoscópica en cuanto a resultados anátomo-patológicos y seguimiento. Material y Métodos: Se realiza un estudio descriptivo revisando registros de pacientes sometidos a linfadenectomía lumboaórtica entre los años 1981 hasta 2003, analizándolos según estadio postoperatorio, recurrencias y evolución clínica. Resultados: La serie consta de 52 pacientes sometidos a linfadenectomía lumboaórtica primaria etapa clínica I. Treinta y una linfadenectomías abiertas (entre 1981-2000) y 21 laparoscópicas (entre 2001-2003). El estudio anátomo-patológico en el grupo total mostró compromiso tumoral ganglionar en 19 pacientes (36,5 por ciento). Lala (+) abierta 10 (32,2 por ciento), Lala (+) laparoscópica 9 (42,8 por ciento). La cantidad total de ganglios obtenidos en Lala abierta fue de 14,6 y en Lala laparoscópica de 11,3 ganglios. El seguimiento promedio fue de 48,4 meses (5-2004), Los pacientes con Lala (+) fueron a quimioterapia y no han presentado recidiva. Los pacientes con Lala (-) abierta 2 han presentado recidiva . El grupo laparoscópico con biopsia negativa no ha registrado recidiva. Conclusión: El manejo de los tumores germinales no seminomatosos con linfadenectomía lumboaórtica demuestra ser efectivo para el diagnóstico de compromiso tumoral retroperitoneal. El valor diagnóstico de linfadenectomía laparoscópica resulta similar a la linfadenectomía abierta.

Glucose transporter (GLUT 4) content and insulin receptor kinase activity in muscles of the LA/N-cp rat.

In this study we compared insulin binding activity, insulin receptor tyrosine kinase activity, and GLUT 4 protein content in six muscles from LA/N-cp rats and their lean controls. LA/N-cp rats had an approximate 20-fold increase in insulin concentration (837 +/- 113 vs 40 +/- 1), associated with significant (p < 0.01) decreases in both insulin binding activity per mg muscle and in muscle GLUT 4 content. Maximum insulin tyrosine kinase activity was also lower in muscle from LA/N-cp rats, but no difference was noted when tyrosine kinase activity was expressed per receptor. These data indicate that there are at least two defects in the insulin action cascade in muscle from LA/N-cp rats that contribute to the insulin resistance in these animals.

The insulin receptor--single function and dual effect.

Net effects of insulin on glucose entry, metabolism and other cellular processes have been well documented over the past 30-40 years. Although it is known that insulin binds to a specific cell membrane receptor protein which undergoes autophosphorylation and tyrosine kinase activation, the individual reactions following receptor activation that cause the metabolic changes remain unknown. It is well documented that the isolated insulin receptor has a high degree of basal autophosphorylation capacity and externally directed tyrosine kinase. There is also evidence that some in vivo autophosphorylation can take place in the total absence of insulin. If receptor activity does exist in the absence of insulin, then receptor function needs to be reanalyzed. It will be proposed here that the insulin binding membrane protein functions mainly to inhibit glucose transport under low physiological levels of insulin. Evidence of basal receptor enzymatic activity in the absence of insulin supports this theory. Under metabolically sufficient conditions, enough insulin receptors are functionally active to interact with the glucose transport system in an inhibitory manner, providing membrane control of internal glucose metabolism. Insulin acts by aggregating this inhibitory system. If inhibitory insulin receptors are aggregated following insulin elevation, their inhibitory action is prevented and glucose transport increases. This increase in transport will be in direct proportion to the temporal inhibitory level of the receptor and to the area of the cell membrane cleared of their inhibitory effect. When insulin receptor protein is confined to small areas of the cell membrane through aggregation, any potential inhibitory function is negated and glucose entry increases dramatically. This is the classical insulin effect. Both of these concepts were suggested 37 years earlier. Randle & Smith (1957, Biochem. Biophys. Acta 25, 442; 1958, Biochem. J. 70, 490) proposed that the internal supply of energy rich compounds limited glucose entry and that the effect of insulin was to inhibit this process which was inhibiting glucose entry. The present report provides a mechanism for this.

Characterization of insulin receptor kinase activity and autophosphorylation in different skeletal muscle types.

We have examined insulin binding, autophosphorylation, and tyrosine kinase activity in detergent-solubilized and wheat germ agglutinin-purified insulin receptor preparations from four rat muscles of different fiber composition (i.e., tensor fascia latae, soleus, vastus intermedius, and plantaris). Insulin binding activity was similar in three of the four muscles but lower in tensor fascia latae. No significant differences were noted in the affinity of insulin for its receptor from various muscle types. Insulin receptor tyrosine kinase activity measured in the absence (basal) and presence of insulin (0.3-300 nM) was comparable in all muscle types (normalized to the amount of insulin bound). Insulin sensitivity, measured as the dose of insulin required for half-maximal activation of kinase activity, was also similar in all muscle types. Likewise, incubation of receptor preparations with [gamma-32P]ATP, Mn2+, and insulin (0.25-100 nM) resulted in a dose-dependent autophosphorylation of the beta-subunit (relative molecular weight approximately 95 kDa) with similar kinetics in all muscle types. In conclusion, these results show that the functional behavior of the insulin receptor autophosphorylation-kinase system (in vitro) is not changed by alterations in muscle fiber composition, indicating that differences in insulin sensitivity between different skeletal muscle types is probably not due to modulation of the insulin receptor phosphorylation system.

Calcium-activated phospholipid-dependent protein kinases from rat liver: characterization of purified isoenzymic forms.

1. This report describes characteristics of the three isoenzymic forms of protein kinase C purified in our laboratory from rat liver. 2. All three C-kinases phosphorylated several histone preparations, and to a much lesser degree, other protein substrates and had similar Km values for ATP and histones. 3. Each isoenzyme demonstrated an absolute requirement for Ca2+ and negatively charged phospholipids. 4. Among various phospholipids tested, phosphatidylserine from bovine brain was most effective with approximately 220 fold stimulation over basal activity. 5. Both diolein and 12-O-tetradecanoylphorbol-13-acetate decreased the Ca2+ requirement of the isoenzymes and also directly stimulated C-II and C-III enzymes in the presence of suboptimal concentrations of Ca2+ and phosphatidylserine.

Identification of isoenzymic forms of hepatic Ca2+-activated-phospholipid-dependent protein kinase in various animal models.

We have examined the protein kinase C that are present in mouse, rat, guinea pig and rabbit liver. Initial subcellular fractionation analysis indicated that the majority (75-85%) of the activity was associated with particulate fraction of the liver. The bound protein kinase C was dissociated by homogenization of livers in buffer containing EGTA, EDTA and various proteolytic inhibitors and the solubilized extract was used to resolve multiple forms of the enzyme. The fractionation procedure, sequentially utilized (NH4)2SO4 precipitation, ion exchange chromatography, gel permeation chromatography, and hydroxylapatite column chromatography. With hydroxylapatite, protein kinase C was resolved into three isoenzymic forms designated C-I, C-II and C-III. In each case, the predominant activity consisted of C-II and C-III and together they represented about 80-88% of the total activity. All three isoenzymes from each source demonstrated an absolute requirement for PS + Ca2+ (approximately 25-50 fold stimulation over basal activity); for maximal activity the isoenzymes also required the presence of divalent metal ion, Mg2+ (5-10 mM) and lysine rich histone (H1). Both diolein and TPA decreased the Ca2+ and PS requirement of the enzyme and directly stimulated enzyme activity in the presence of suboptimal concentrations of Ca2+ and PS. In conclusion, the present studies suggest that protein kinase C in mammalian liver exists in isoenzymic forms.

Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms.

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.

The influence of age on steroidogenic enzyme activities of the rat adrenal gland: enhanced expression of cholesterol side-chain cleavage activity.

The ability of isolated adrenocortical cells to secrete corticosterone in response to ACTH challenge declines as rats age, but the site or mechanism(s) of this impairment is still unknown. To test the functionality of steroidogenic capacity per se, we measured the key enzyme activities involved in corticosterone biosynthesis. We also measured the mitochondrial cytochrome P-450 content and nonsteroidogenic enzymes specific for subcellular fractions. Mitochondria and microsomal fractions were isolated from the adrenals of 2-, 12-, and 18-month-old animals and used for various enzyme measurements. Mitochondrial side-chain cleavage enzyme activity (nanomoles per min mg protein-1) increased from a mean of 0.43 +/- 0.06 in 2-month-old rats to 1.26 +/- 0.11 and 1.51 +/- 0.06 in 12- and 18-month old rats, respectively. After incubation with 5-cholesten-3 beta,25-diol (25-hydroxycholesterol; 25 micrograms/ml) side-chain cleave activity rose to 5.0 +/- 0.6, 12.4 +/- 1.2, and 16 +/- 1.4 nmol min-1 mg protein-1 in adrenal mitochondrial fractions from 2-, 12-, and 18-month-old rats, respectively. In contrast, mitochondrial cytochrome P-450 content did not vary with advancing age. Microsomal delta 5-3 beta-hydroxysteroid dehydrogenase-delta 5-delta 4-isomerase activities were similar in 2- and 12-month-old rats, but 21-hydroxylase (nanomoles per min mg protein-1) activity was significantly increased in 12-month-old rats (2-month-old, 5.2 +/- 0.2; 12-month-old, 7.7 +/- 0.5). Finally, mitochondrial 11 beta-hydroxylase was comparable in both age groups. In addition, activities of mitochondrial nonsteroidogenic enzymes, such as monoamine oxidase, amytal insensitive NADH cytochrome c reductase, cytochrome c oxidase, succinate dehydrogenase, and malate dehydrogenase, did not change with age. It appears from the evidence presented that the activities of the steroidogenic enzymes are not responsible for the diminished capacity in corticosterone production seen with aging in the rat.

Endocrine effects of meternal alcoholization: plasma and brain testosterone, dihydrotestosterone, estradiol, and corticosterone.

Maternal ethanol consumption was associated with reduced levels of dihydrotestosterone in the brains of 1--2-day-old male rats when compared to those of sex-matched pups obtained from dames that were fed sucrose. In contrast, brain levels of corticosterone were increased significantly in the pups of ethanol-fed animals when compared to those from sucrose-fed controls. Brain and plasma estradiol did not differ between groups. These results suggest that maternal ethanol consumption may influence the central nervous system and plasma levels of certain steroidal hormones in the offspring.

Brain corticosterone in lactating rats: possible relation to the attenuation of the pituitary-adrenocortical response to stress.

In an attempt to elucidate the mechanism of suppression of the plasma corticosterone stress response in the lactating female rats, nonstress levels of corticosterone in plasma and hypothalamus, cerebral cortex, hippocampus and pituitaries were determined in the lactating and nonlactating Sprague-Dawley rats at 1600-1700 hr by the protein binding assay. The brain corticosterone levels were significantly higher (64-92%) in the lactating than in nonlactating rats, while the plasma corticosterone levels were identical. We conclude that the suppression of the stress response in the lactating female rats is directly or indirectly related to the high basal levels of corticosterone in the brain, which result from a chronic hyperactivation of the endocrine axis.

The fluorometric assay of rat plasma corticosterone: evaluation of nonspecific fluorescence.

An analysis of 168 plasma samples from intact rats, one to 35 days of age, was performed using both brief and specific fluorometric procedures. The amount of fluorescence produced by the brief procedure which could be attributed to corticosterone ranged from a maximum of 72% to a minimum of 16% of the total fluorescence value. Corticosterone represented 50% or more of the brief assay value in only five out of 18 groups of animals assayed. Following statistical analysis of the nonspecific fluorescence, a significant variation was found due to the age of the animal. A highly significant increase in nonspecific fluorescence was found in 21-day old animals following histamine injection. It was concluded that the brief fluorometric assays for corticosterone were of little value if specificity was desired.

Relationship of intracellular creatine concentration and uptake to muscle mass in vivo.

Attempts have been made to evaluate the role of intracellular creatine in conditions leading to increased or decreased amounts of contractile protein in rat skeletal muscles. Resting concentrations of intracellular creatine ([Cr]i) and creatine phosphate ([CrP]i) were compared in gastrocnemius and soleus muscles with those immediately after a 20-s tetanic stimulation. The hydrolysis of creatine phosphate was the same after heavily and lightly loaded contractions, suggesting that hypertrophy of isometric exercise is not mediated by creatine. With atrophy after denervation or interruption of sciatic axoplasmic flow [Cr]i also remained unchanged, though [CrP]i and the rate of Cr uptake fell after denervation. The major change in adult red and white muscle bulk with unaltered [Cr]i suggests that the Cr sensitivity found by others in developing muscle in vitro has been supplemented or replaced by other control mechanisms.

Circadian rhythm of corticosterone levels in rat brain.

In the present study the circadian changes which occur in the levels of corticosterone in the brain and plasma in Sprague-Dawley rats are reported. The levels of corticosterone in the brain were found to have a daily trough and crest with timing similar to that observed for the plasma steroid. In addition, the effect of histamine stress on the corticosterone content of the particulate and the soluble fractions at the trough and crest was examined. The levels of both brain fractions were significantly higher 20 min after histamine injection. The time of day at which the stress was applied was not a significant factor in the magnitude of the stress response.