The hypothesis that modifications in beta-adrenergic receptors may be responsible for age-dependent change previously observed in vivo, has been investigated. Beta-adrenoceptor characteristics of submandibular glands of mice were studied by using the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Data from such studies indicated the presence of two functional populations of binding sites in membrane preparations from young animals, displaying high and low affinity, respectively. Experiments performed on old mice membrane preparations revealed a 50% decrease in the high-affinity population receptor number when compared to the preparations from young animals. However, the affinity did not change significantly with advancing age. With regard to the low-affinity population, no statistically significant changes were observed. From these data it can be reasonably assumed that beta-adrenoceptor alteration during ageing may play a major role in the age-dependent impairment of beta-adrenergic responses in vivo.

Aging↗

PubMedDate not supplied

Food restriction in female Wistar rats. I. Survival characteristics, membrane microviscosity and proliferative response in lymphocytes.

The effect of food restriction on the survival characteristics, membrane microviscosity and proliferative response in lymphocytes of female Wistar undernourished rats has been evaluated. Diet restriction was applied starting from the age of 3.5 months by feeding the animals on an every-other-day schedule (EOD). Diet restricted animals showed an increase of both mean, median and maximal life span as compared to the rats fed ad libitum (AL). Analyzing the survival curves by a parametric model, it emerged that undernutrition increased the individual resistance to environmental insults. In particular, it could be speculated that the positive influence was more pronounced in individuals with the lowest physiological capacities. The membrane microviscosity of lymphocytes was lower in EOD animals as compared to the AL ones even if one assumes a decrease in body temperature of 1-2 degrees C in EOD groups. The improvement of membrane microviscosity due to diet restriction may in part explain the improvement of proliferative response of lymphocytes from EOD groups.

Journal Article↗

PubMedDate not supplied

Functional interrelationships in aging processes: alterations and reversibility.

The problem of assessing the relevance of the reversibility of age-related functional alterations for aging studies has been presented. Transduction mechanisms of adrenergic stimulation have been chosen as the target of age-related changes and thymus as the effector of some corrective interventions performed at advanced age. Both alterations of adrenoceptor characteristics and their reversibility have been reviewed. beta-adrenoceptors have been studied in organs bearing only one subtype of receptors or both, revealing an age-related decrease in density only in the beta1-subtype. It has been shown that a similar age-related decrease is present in alpha1-adrenoceptor density. Such alterations are corrected by grafting a neonatal thymus into old mice. On the contrary, thymus fails to correct the alteration of T4-induced upregulation of beta-adrenoceptors indicating some limits to its corrective effect when the net of functional interrelationships becomes relatively complex. Both failures and successes of thymic grafts and thymic extracts in reversing age-related changes are discussed taking into account the effects induced on the life span of the animals. Different unsolved problems stemming from the previous considerations are also presented. Among them the controversial question about linearity and non-linearity of biological parameters presumed to be good indices of aging is discussed, with the aid of a simple model as a schematic example.

Journal Article↗

PubMedDate not supplied

Parallel decrease of adenylyl cyclase activity and beta1-adrenoceptor density in brain cortex of aging mice.

Beta-adrenergic receptors (betaARs) and adenylyl cyclase (AC) activity have been found to undergo progressive alterations in the brain cortex of aging mice. In particular, betaAR changes are in charge of the beta1 subpopulation (beta1AR), beta2-adrenoceptors (beta2ARs) showing no age-related impairments. On these bases, the question arises whether AC alterations can be accounted for by beta1AR changes or, alternatively, whether there are also receptor independent AC modifications. Experiments have thus been performed on the brain cortex from young and old mice assaying beta1AR and beta2AR characteristics and isoproterenol (IPR) and forskolin stimulated AC activity in the same membrane preparations. Other membranes were previously treated with the reducing agent dithiothreitol (DTT) which is known to specifically destroy the binding capacity of beta1ARs. No statistically significant differences in basal AC activity have been found among the groups studied. Data on IPR-stimulated AC activity in old animals confirm previous findings demonstrating impaired responsiveness when compared with that of young mice. DTT treated membrane preparations from both young and old mice show decreased AC activities. The entity of the decrease is lower in aged animals due to the lower initial level of beta1ARs. Forskolin stimulation, which is assumed to directly activate AC, has been found impaired in aged mice when compared with young animals, suggesting that also post-receptor alterations occur with advancing age.

Journal Article↗

PubMedDate not supplied

Alterations of brain insulin receptor characteristics in aging mice.

An interesting role of insulin and insulin receptors (InsRs) in the brain is neuromodulation of monoaminergic systems. Since our previous studies showed age-dependent alterations of alpha- and beta-adrenoceptors in mouse brain cortex, the intriguing role of brain InsRs per se and their involvement in adrenergic modulation prompted us to check their eventual changes with aging. Thus, brain InsR characteristics were studied in young (3 months) and old (27 months) Balb/c-nu mice by direct binding with (125)I-insulin. A two-sites model analysis of data shows a statistically significant age-related decrease of receptor density (39%) and k(d) (57%) in the high affinity population. The low affinity receptor subset also shows a decreasing trend of its characteristics; however, differences are not statistically significant and show a high degree of interindividual variability in both groups of mice.

Journal Article↗

PubMedDate not supplied

Excitatory amino acid receptors in the prefrontal cortex of aging mice.

The zones of the prefrontal cortex of Balb/c mice were tested for age-related changes of the ionotropic excitatory amino acid receptors density, together with zones of the dorsal cortex. Kainate, N-methyl-D-aspartate, and amino-3-hydroxy-5-methyloxazole-4-propionate sites were measured by slice receptor binding techniques in cortical zones from animals at the age of 6, 12, 18, and 24 months. An increase of the N-methyl-D-aspartate sites was detected in the medial prefrontal zone of mid-aged animals and was followed by a decrease at old age; a decrease of the N-methyl-D-aspartate and kainate sites was found for the medial dorsal (cingulate) cortex at old age. The age-related changes of receptor densities in the different cortical areas seem unrelated in origin. The sites decrease in the cingulate cortex could affect the transfer of the prefrontal cortex activity toward limbic structures.

Aging↗

PubMedDate not supplied

Modulation of mouse brain cortex adrenoceptor in old mice by supplementation of zinc and thymomodulin.

BACKGROUND: Previous experiments have shown that the age-related decrease of mouse brain cortex adrenoceptor density can be recovered by grafting a neonatal thymus into old recipients. The question arises whether similar results can also be obtained in the presence of a single thymic factor such as thymomodulin (TMD). It is worth noting that the activity of some thymic factors is strictly zinc (Zn) dependent and that their age-related decreased production is recovered in old mice supplemented with Zn. OBJECTIVE: The above-mentioned evidences prompted us to verify whether Zn and TMD, either alone or combined, are able to induce some corrective effects on age-dependent alterations of adrenoceptor characteristics of the mouse brain cortex. METHODS: Thus, we performed experiments on four groups of Balb/c mice treated with saline, Zn, TMD, or both Zn and TMD. Treatments started when animals were 18 months old and ran for 6 months. The alpha(1)- and beta-Adrenoceptor characteristics were assayed by steady state binding analysis using labelled prazosine and iodocyanopindolol, respectively. Data were analyzed using one-way analysis of variance, followed, when appropriate, by multiple-comparison analysis. RESULTS: Results show an increase of beta-adrenoceptor density and a decrease of alpha(1)-adrenoceptor density in both Zn- and Zn+TMD-treated animals when compared to saline-treated controls, while receptor affinities did not change significantly. CONCLUSIONS: The lack of action of TMD suggests that this type of treatment cannot mimic the effects of grafting the whole gland; it cannot be excluded that different time-dose schedule could be more effective. Zn treatment, on the other hand, does modulate adrenoceptors; however, it shows a corrective action on the age-related decreased density of beta-adrenoceptors, but further decreases that of alpha(1)-adrenoceptors. This differential action could be due to their differential physiological role.

Aging↗

PubMedDate not supplied

Transcriptional regulation of the androgen signaling pathway by the Wilms' tumor suppressor gene WT1.

The androgen-signaling pathway plays a critical role in prostate cancer development and progression. We have recently demonstrated that the Wilms' tumor suppressor gene product, WT1, binds to multiple sites in the androgen receptor (AR) promoter and transcriptionally represses the AR gene promoter in vitro. We asked whether WT1 repression of the endogenous AR gene interferes in the androgen signal transduction cascade and modifies AR target gene expression. We analyzed the effect of WT1 (-/-) overexpression on an AR target gene reporter construct that contains the luciferase gene, the ElB TATA box, and two copies of the androgen-response element (ARE), the dimeric AR binding site. Luciferase activity was determined in 293 kidney and TM4 Sertoli cells, two nontumorigenic cell lines that express both AR and WT1. Cells were cotransfected by lipofectamine in the presence or absence of the synthetic androgen R1881. Results showed that overexpression of WT1 downregulates ARE-reporter gene transcription in both cell lines tested. The inhibitory effect of WT1 on the AR target gene construct was dose-dependent and androgen-independent in 293 cells, whereas in TM4 cells it was androgen-dependent. Additionally, a zinc-finger mutant WT1 (-/-) expression construct, R394W, failed to decrease luciferase activity, suggesting that WT1 downregulates the ARE-reporter gene construct activity by directly repressing the endogenous AR gene promoter. Furthermore, we analyzed the expression of WT1 and AR mRNA in several prostate cancer cell lines in order to understand the role WT1 may play in prostate cancer development and progression. Gel analysis of cDNA amplified by RT-PCR of AR and WT1 RNA from prostate cancer and non-prostatic cell lines showed that LNCaP and MDAPCa2b, two metastatic prostate cancer cell lines which are androgen-sensitive, expressed AR but not WT1. Du145 and PC3, two cell lines from advanced metastatic prostate cancer, which are characterized as androgen-independent and -insensitive, did not express AR but expressed a high level of WT1. Two non-prostatic cell lines, T47D and 293, weakly co-expressed AR and WT1. This inverse relationship between AR and WT1 expression in prostate cancer cell lines, together with WT1 repression of the AR promoter, suggest a role for WT1 in the androgen signaling pathway and in prostate cancer development and progression.

Androgens↗

PubMedDate not supplied

Blood catecholamine levels and lymphocyte beta-adrenoceptors following acute noise stress.

Blood hormonal levels and lymphocyte beta-adrenoceptor characteristics have been studied in subjects exposed to acute noise stress. Cortisol and DHEAS show a peak at 15 min after the beginning of the stimulus. Catecholamine levels show an increase in 4 out of 8 subjects. However, no statistically significant changes have been observed in beta-adrenoceptor characteristics.

Catecholamines↗

PubMedDate not supplied

Assay of estrogen and progesterone receptors in small samples of breast cancer: I. Mathematical analysis of total binding data.

The quantitative determination of Estrogen and Progesterone receptors in breast cancer is performed using about 500 mg of tissue. Such an amount of tissue sample is available with ever increasing difficulty. In the present paper a mathematical method of analysis of the total (specific+nonspecific) binding data is given. In this way about 50% of tissue can be saved as only one set of test tubes is required; in fact, the method does not need the second set of tubes used to determine nonspecific binding in the conventional assay. Additional advantages from the statistical point of view are also presented.

Animals↗

PubMedDate not supplied

Assay of estrogen and progesterone receptors in small samples of breast cancer: II. The conventional procedure compared to the mathematical one.

Parallel assays of ER and PR receptors have been performed in the same tissue samples using both the conventional method employing two sets of test tubes and a mathematical method which does not need the second set of tubes containing excess cold ligand concentrations. Determinations have been performed in calf uterus and human breast cancer samples. Findings show that the two methods give quite similar results. In particular, they are very close in human BC tissues where receptor density values show a very wide range. The mathematical method seems quite useful when the samples are poor of receptors, a case where the precision of the method may influence both prognosis and therapy.

Animals↗

PubMedDate not supplied

Determination of the optimal conditions to study DNA synthesis in cultures of mouse hepatocytes.

Mouse hepatocytes undergo profound changes during aging, such as increased nuclear volume, occurrence of tetraploid nuclei and impaired DNA synthesis following in vivo adrenergic stimulation. These alterations have been found reversible by implanting a neonatal thymus into old animals. In the present paper we studied the optimal experimental conditions in order to investigate the mechanisms of such actions in vitro. Unfortunately, in fact, most of data in the field comes from experiments on rats. Thus, we examined DNA synthesis of hepatocytes from mice cultivated in presence of neonatal calf serum only, or after the addition of the beta-adrenergic agonist isoproterenol or the beta-adrenergic antagonist propranolol. DNA synthesis in hepatocytes from young Balb/c-nu mice shows a peak between 36 and 48 h even in the absence of any specific stimulation other than newborn calf serum. The addition of isoproterenol does not modify the DNA synthetic pattern, while propranolol causes a slight but statistically significant decrease in 3H-thymidine incorporation. Results are compared with those obtained from other authors in rats.

Adrenergic beta-Agonists↗

PubMedDate not supplied

Assay of insulin receptors in mouse brain.

We previously demonstrated that mouse adrenergic system undergoes age-related impairments which can be reversed by grafting thymus into old animals. Recently, our attention is devoted to brain insulin receptors (InsRs), because of their possible involvement in neuromodulation of monoaminergic systems. The paucity of information on brain InsRs in general, and on mouse in particular, prompted us to look for methods by which brain InsR characteristics can be determined accurately, before beginning a study on possible age-dependent modifications of this receptor system and their eventual recovery by thymus graft. Brain insulin receptor characteristics were studied in a group of young Balb/c-nu mice by binding competition experiments, set up incubating fresh brain membranes with a constant amount of 125I-insulin in presence of increasing concentrations of cold insulin. Experimental data were analysed using both one-site and two-site models. Comparison of results demonstrates that curvilinear Scatchard plot of brain InsRs is indicative of the presence of two binding sites with high and low affinity, respectively. Data also shows that density and affinity of the high affinity receptor subset can be determined accurately, while the low affinity receptor subpopulation presents a high degree of interindividual variability for both density and affinity. It can be concluded that this method of determination of InsR characteristics can be safely used to deepen the study of thymus graft-induced recovery of age-related modifications of brain InsR system.

Animals↗

PubMedDate not supplied

Assay of total protein kinase activity in mouse brain cortex.

In previous studies we demonstrated that the adrenergic system is impaired in old animals and that the main alterations were observed at the level of receptor density and adenylate-cyclase activity. The decreased ability to produce cAMP could influence the activity of the cAMP dependent protein kinase (PKA), one of the enzymes responsible for the phosphorylation of protein substrates. Since protein phosphorylation is one of the most common and important mechanisms through which a cell regulates its activity, the characteristics of the phosphorus incorporation reaction were studied. Kinase activity was measured in homogenate of young mouse brain cortex prepared avoiding gross manipulations in order to maintain conditions as close to those present in the living animal as possible. Results show that phosphate incorporation is proportional to protein content and strictly dependent on ATP availability. Increasing the ATP concentration from 10 to 500 mumol/l, the length of incorporation phase increases, suggesting that the limiting point of the reaction is better represented by energy availability than by enzyme or protein substrate concentrations.

Adenosine Triphosphate↗