The subcellular distribution of glucocorticoid-receptor complexes as studied by chemical crosslinking of intact HTC cells

Treatment of intact HTC cells with glutaraldehyde results in redistribution of glucocorticoid binding sites between cytosolic and nuclear fractions. The decrease in cytosolic receptors and their accumulation at the nuclear level were found to be directly related to the glutaraldehyde concentrations employed in our procedure and inversely related to the cell density of samples. When the data from eleven separate experiments were combined, and analyzed by linear regression of cytosolic and nuclear levels of receptor complexes vs the ratios between the DNA and glutaraldehyde concentration of our samples, two lines were obtained whose intercepts on the ordinate yielded values of cytosolic and nuclear receptors corresponding to 37.5 and 62.5% of the total cellular pool, respectively. When we compared the subcellular redistribution of glucocorticoid receptor to that of the cytosolic enzyme lactate dehydrogenase upon HTC cell crosslinking with glutaraldehyde, we found that the cytosolic and nuclear levels of the enzyme were 53.2 and 46.8% of the total content, respectively. If the subcellular distribution of glucocorticoid receptor is corrected for the artefactual redistribution induced by crosslinking, using the values obtained for lactate dehydrogenase, it can be concluded that glucocorticoid receptors in HTC cells are distributed between cytosol and nuclei in a ratio which is about 2:1. Our findings lend further support to theconclusion that only a portion of glucocorticoid receptor is cytosolic in intact cells.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone     

Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Release of cholecystokinin in the central nervous system

The octapeptide cholecystokinin (CCK) is one of the most abundant neuropeptides of the central nervous system. A number of features (for instance heterogeneity of the regional distribution, subcellular localization at the nerve terminal level, calcium-dependent release upon nervous tissue depolarization) support the candidacy of CCK as a neurotransmitter. The reported co-existence of CCK and dopamine in some meso-limbic neurons has led to speculation that the neuropeptide may interact with the catecholamine in neuropsychopathologies linked to dopamine dysfunctions, like schizophrenia. Data from the experimental animals have so far generated conflicting results. It should be noted that the interactions between CCK and dopamine, and, in particular, the effects of CCK and dopamine on each other release, both in vitro and in vivo, have been poorly investigated and would require special attention. Evidence is accumulating that CCK may participate in the expression of anxiety. Indeed antagonists at the central CCK receptors exhibit anxiolytic activity in the laboratory animal. An interesting linkage appears to exist in the brain between 5-hydroxytryptamine (5-HT) and CCK. Activation of 5-HT₃ receptors was found to increase CCK release from rat cortical or nucleus accumbens synaptosomes. Interestingly, antagonists at 5-HT₃ receptors appear to possess anxiolytic activity. Recent studies carried out in conscious unrestrained rats show that the calcium-dependent, tetrodotoxin-sensitive release of CCK-like immunoreactivity evoked in the rat frontal cortex by veratrine infusion can be inhibited by submicromolar concentrations of 5-HT₃ receptor antagonists. It seems legitimate to conclude that 5-HT and CCK interact in the living brain, the former increasing the release of the latter through activation of receptors of the 5-HT₃ type. On the basis of this interaction both 5-HT₃ and CCK receptor antagonists may become novel anxiolytics.     

Rhythmical timing and spatial scattering of foraging in a homer limpet {Patella rustica)

Homing to a more or less permanent scar after each foragingexcursion is a common movement pattern among intertidal gastropodsand chitons; however, details of the timing and spacing of foragingactivity have been investigated only in a few species. The presentstudy analyzes the short-term behavior of the limpet Patellarustica along the Tyrrhenian coast, Italy, using a motographictechnique to assess the fine organization of its foraging duringfavorable periods of sea roughness. P. rustica becomes activeonce the upper midlittoral is well splashed. It alternates foragingexcursions and resting at home with a periodicity slightly longerthan 12 h, suggesting a tidal-diel pattern. However, periodogramanalysis of the sea level oscillations during the study periodsrevealed no such rhythmicity because tidal oscillations werehidden by irregular variations caused by waves. As a resultof this time partitioning, limpets move, on average, less than50% of their potential activity time. Time partitioning maybe highly adaptive in reducing potential risks. Nevertheless,in the absence of clear external driving cues, the significanceof a very regular and apparently tidal pattern, fairly synchronousamong the different specimens, remains to be explained. Theactivity of P. rustica during each excursion is organized intothree parts: the outgoing journey during which grazing activityprogressively increases, a central part characterized by intensegrazing, and the return characterized by fast displacement anda more or less consistent trail following. Limpets head forrandom directions to reach foraging grounds in successive excursions,showing only a slight avoidance of the direction taken duringthe previous outward journey. This pattern produces a spatialscattering of grazing activity, allowing efficient exploitationof grazing areas distributed radially around home during subsequentexcursions.     

A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

Mechanisms of group-hunting in vertebrates

Group-hunting is ubiquitous across animal taxa and has received considerable attention in the context of its functions. By contrast much less is known about the mechanisms by which grouping predators hunt their prey. This is primarily due to a lack of experimental manipulation alongside logistical difficulties quantifying the behaviour of multiple predators at high spatiotemporal resolution as they search, select, and capture wild prey. However, the use of new remote-sensing technologies and a broadening of the focal taxa beyond apex predators provides researchers with a great opportunity to discern accurately how multiple predators hunt together and not just whether doing so provides hunters with a per capita benefit. We incorporate many ideas from collective behaviour and locomotion throughout this review to make testable predictions for future researchers and pay particular attention to the role that computer simulation can play in a feedback loop with empirical data collection. Our review of the literature showed that the breadth of predator:prey size ratios among the taxa that can be considered to hunt as a group is very large (<10⁰ to >10²). We therefore synthesised the literature with respect to these predator:prey ratios and found that they promoted different hunting mechanisms. Additionally, these different hunting mechanisms are also related to particular stages of the hunt (search, selection, capture) and thus we structure our review in accordance with these two factors (stage of the hunt and predator:prey size ratio). We identify several novel group-hunting mechanisms which are largely untested, particularly under field conditions, and we also highlight a range of potential study organisms that are amenable to experimental testing of these mechanisms in connection with tracking technology. We believe that a combination of new hypotheses, study systems and methodological approaches should help push the field of group-hunting in new directions.     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Genetic metabolic polymorphisms and the risk of cancer: a review of the literature

The purpose of this paper is to systematically analyse the design and results of epidemiological studies on the association between various types of cancer (lung, bladder, breast, colon, stomach) and four genetically-based metabolic polymorphisms, involved in the metabolism of several carcinogens (glutathione-S-transferase M1, debrisoquine hydroxylase, N acetyltransferase, aryl hydrocarbon hydroxylase). These inherited polymorphisms usually cause modifications in the quality or quantity of the relevant enzymes. Such enzymes are involved in the activation/inactivation of known carcinogens and seem to modify the extent to which carcinogens interact with DNA in target tissues. Two enzymes, debrisoquine hydroxylase and aryl hydrocarbon hydroxylase, activate procarcinogens to carcinogens (phase I enzymes). The other two, glutathione-S-transferase M1 and N-acetyltransferase, mainly detoxity carcinogenic substances (phase II enzymes). Because of their role as host factors (modulating the action of carcinogens), it has been hypothesized that subjects presenting a specific phenotype for such polymorphisms could be at a greater risk of developing various types of cancer. A number of epidemiological studies have investigated such associations, often with discordant results. We examine and discuss the design of the studies, and present a meta-analysis of the available data.