Chronobiological study of free thyroid hormones in hypothyroid and hyperthyroid subjects

Previous reports demonstrate a circadian rhythm of the free thyroid hormones in healthy subjects. In this study we evaluated circadian variation of FT3 and FT4 in hyperthyroid and hypothyroid states. We considered six hyperthyroid patients, six hypothyroid patients and six control subjects. Blood samples were taken two hours apart from a catherterized arm vein. Data were evaluated by Halberg's cosinor analysis. The results show that FT3 and FT4 exhibit a circadian rhythm in healthy subjects, not evident in hyperthyroid and hypothyroid patients.     

Chronobiological Aspects of Weight Loss in Obesity: Effects of Different Meal Timing Regimens

A series of short- and long-lasting experimental protocols of different meal timing regimes were performed in obese subjects to assess the possible occurrence of (1) a different metabolic fate of nutrients; (2) a phase shift of circadian rhythms of metabolic and hormonal parameters strictly related to nutrition; (3) a different weight loss. (A) In a short-lasting protocol (3 days) IS obese subjects were fed a hypocaloric diet (684 kcal/day) (a) at 10 hr only, (b) at 1800 hr only; (c) at 1000 hr, 1400 hr and 1800 hr, or (d) studied during a 36-hr fasting. Measures of calorimetry (R.Q., CHO and lipid oxidations, energy expenditure), hormones (plasma Cortisol, insulin, HGH, urinary catecholamines), urinary electrolytes (Na, K) and vital parameters (body temperature, heart rate, blood pressure) were carried out at 4-hr intervals for three days. A significantly higher lipid oxidation and a lower CHO oxidation were documented with the meal at 1800 hr, in comparison with the meal at 1000 hr. CHO and lipid oxidation circadian rhythms appeared the most affected by meal timing. (B) In a long-lasting protocol (18 days) 10 obese subjects were fed the same hypocaloric diet (a) at 1000 hr only and (b) at 1800 hr only. Calorimetric measures were performed every other day for 2 hr preceding each meal. Before and after the 18-days single meal period, body temperature, plasma Cortisol, PRL and TSH were recorded (δt = 4 hr). A higher lipid oxidation and a lower CHO oxidation were again demonstrated with the meal at 18 hr. Minimal changes of hormonal circadian rhythms were documented suggesting that the hypothalamus-hypophysis network is scarcely affected by meal timing. Weight loss did not vary in both short- and long-term protocol.     

Differential loss of T cell signaling molecules in metastatic melanoma patients' T lymphocyte subsets expressing distinct TCR variable regions

In this study we tested the hypothesis that loss of T cell signaling molecules in metastatic melanoma patients' T cells may affect differently T cell subsets characterized by distinct TCR variable regions. By a two-color immunofluorescence technique, expression of zeta-chain, lck, and ZAP-70 was evaluated in CD3+ T cells and in three representative T cell subsets expressing TCRAV2, TCRBV2, or TCRBV18. Partial loss of lck and ZAP-70 was found in CD3+ T cells from PBL of most melanoma patients, but not of healthy donors. The extent of zeta-chain, lck, and ZAP-70 loss depended on the TCRV region expressed by the T cells, and this association was maintained or increased during progression of disease. Coculture of patients' or donors' T cell with melanoma cells, or with their supernatants, but not with normal fibroblasts or their supernatants, down-modulated expression of zeta-chain, lck, and ZAP-70 in a TCRV region-dependent way. Immunodepletion of soluble HLA class I molecules present in tumor supernatants, but not of soluble ICAM-1, blocked the suppressive effect on T cell signaling molecule expression. T cell activation with mAbs to a single TCRV region and to CD28 led to significant and TCRV region-specific re-induction of zeta-chain expression. These findings indicate that extent of TCR signaling molecules loss in T lymphocytes from metastatic melanoma patients depends on the TCRV region and suggest that tumor-derived HLA class I molecules may contribute to induce such alterations.     

Measuring zinc in living cells. A new generation of sensitive and selective fluorescent probes

New fluorescent indicators with nanomolar to micromolar affinities for Zn(2+) have been synthesized in wavelengths from UV to the far red. The UV light-excited indicators are ratiometric. The visible wavelength indicators are non-ratiometric and exhibit large and pH-independent fluorescence increases with increasing zinc concentrations, with little to no sensitivity to physiologically relevant Ca(2+) concentrations. Experiments in neuronal and non-neuronal cell cultures show the new indicators to retain their sensitivity to and selectivity for zinc after conversion to cell-permeable forms.     

Rethinking the excitotoxic ionic milieu: the emerging role of Zn(2+) in ischemic neuronal injury

Zn(2+) plays an important role in diverse physiological processes, but when released in excess amounts it is potently neurotoxic. In vivo trans-synaptic movement and subsequent post-synaptic accumulation of intracellular Zn(2+) contributes to the neuronal injury observed in some forms of cerebral ischemia. Zn(2+) may enter neurons through NMDA channels, voltage-sensitive calcium channels, Ca(2+)-permeable AMPA/kainate (Ca-A/K) channels, or Zn(2+)-sensitive membrane transporters. Furthermore, Zn(2+) is also released from intracellular sites such as metallothioneins and mitochondria. The mechanisms by which Zn(2+) exerts its potent neurotoxic effects involve many signaling pathways, including mitochondrial and extra-mitochondrial generation of reactive oxygen species (ROS) and disruption of metabolic enzyme activity, ultimately leading to activation of apoptotic and/or necrotic processes. As is the case with Ca(2+), neuronal mitochondria take up Zn(2+) as a way of modulating cellular Zn(2+) homeostasis. However, excessive mitochondrial Zn(2+) sequestration leads to a marked dysfunction of these organelles, characterized by prolonged ROS generation. Intriguingly, in direct comparison to Ca(2+), Zn(2+) appears to induce these changes with a considerably greater degree of potency. These effects are particularly evident upon large (i.e., micromolar) rises in intracellular Zn(2+) concentration ([Zn(2+)](i)), and likely hasten necrotic neuronal death. In contrast, sub-micromolar [Zn(2+)](i) increases promote release of pro-apoptotic factors, suggesting that different intensities of [Zn(2+)](i) load may activate distinct pathways of injury. Finally, Zn(2+) homeostasis seems particularly sensitive to the environmental changes observed in ischemia, such as acidosis and oxidative stress, indicating that alterations in [Zn(2+)](i) may play a very significant role in the development of ischemic neuronal damage.     

Zn(2+) induces permeability transition pore opening and release of pro-apoptotic peptides from neuronal mitochondria

Rapid entry of Ca(2+) or Zn(2+) kills neurons. Mitochondria are major sites of Ca(2+)-dependent toxicity. This study examines Zn(2+)-initiated mitochondrial cell death signaling. 10 nm Zn(2+) induced acute swelling of isolated mitochondria, which was much greater than that induced by higher Ca(2+) levels. Zn(2+) entry into mitochondria was dependent upon the Ca(2+) uniporter, and the consequent swelling resulted from opening of the mitochondrial permeability transition pore. Confocal imaging of intact neurons revealed entry of Zn(2+) (with Ca(2+)) to cause pronounced mitochondrial swelling, which was far greater than that induced by Ca(2+) entry alone. Further experiments compared the abilities of Zn(2+) and Ca(2+) to induce mitochondrial release of cytochrome c (Cyt-c) or apoptosis-inducing factor. In isolated mitochondria, 10 nm Zn(2+) exposures induced Cyt-c release. Induction of Zn(2+) entry into cortical neurons resulted in distinct increases in cytosolic Cyt-c immunolabeling and in cytosolic and nuclear apoptosis-inducing factor labeling within 60 min. In comparison, higher absolute [Ca(2+)](i) rises were less effective in inducing release of these factors. Addition of the mitochondrial permeability transition pore inhibitors cyclosporin A and bongkrekic acid decreased Zn(2+)-dependent release of the factors and attenuated neuronal cell death as assessed by trypan blue staining 5-6 h after the exposures.     

In vitro short-term test evaluation of catecholestrogens genotoxicity

Estrogens are indicated as being the most important etiological factors for the development and progression of breast cancer. The implication of estrogen in breast cancer has been associated mostly with the estrogen receptors that mediate cell proliferation. Evidence also exists to support the hypothesis of a direct role of estrogens as tumor initiators. However, the role of estrogen genotoxicity in breast cancer is still questionable.

In this study the genotoxic activity of catecholestrogens and 16-hydroxy estrone has been investigated by performing Salmonella strain TA98 and TA100 Ames tests, sister chromatide exchange assays (SCE) and micronucleus assays on human peripheral lymphocytes (CBMN and ARA/CBMN). We found a lack of positive results with micronucleus assays, except for 2-hydroxy estradiol (2-OHE₂), which shows a peculiar “bell shaped” trend of micronucleus number versus concentrations. SCE assay suggests weak genotoxic activity of all tested catechol metabolites, except 4-hydroxy estrone (4-OHE₁), which also showed negative results by ARA/CBMN.

In this open debate, our results support the hypothesis of a weak genotoxicity, not correlated with the carcinogenetic potential of estrogens.     

A computational modelling approach to investigate different targets in deep brain stimulation for Parkinson’s disease

We investigated by a computational model of the basal ganglia the different network effects of deep brain stimulation (DBS) for Parkinson’s disease (PD) in different target sites in the subthalamic nucleus (STN), the globus pallidus pars interna (GPi), and the globus pallidus pars externa (GPe). A cellular-based model of the basal ganglia system (BGS), based on the model proposed by Rubin and Terman (J Comput Neurosci 16:211–235, 2004), was developed. The original Rubin and Terman model was able to reproduce both the physiological and pathological activities of STN, GPi, GPe and thalamo-cortical (TC) relay cells. In the present study, we introduced a representation of the direct pathway of the BGS, allowing a more complete framework to simulate DBS and to interpret its network effects in the BGS. Our results suggest that DBS in the STN could functionally restore the TC relay activity, while DBS in the GPe and in the GPi could functionally over-activate and inhibit it, respectively. Our results are consistent with the experimental and the clinical evidences on the network effects of DBS.