Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: A comparative study on the effects of coagulant and non-coagulant fixatives

Summary The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulatn fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.     

Biosynthesis of the sesquiterpenoid ageratriol

Ageratriol is biosynthesized from agerol through a diepoxide derivative. Mevalonic acid incorporation revealed that the formation of the isopropenylic double bond is not stereospecific.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Comparison of multiple molecular dynamics trajectories calculated for the drug-resistant HIV-1 integrase T66I/M154I catalytic domain

HIV-1 integrase (IN) is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. Single and multiple mutations of IN at residues T66, S153, or M154 confer degrees of resistance to several inhibitors that prevent the enzyme from performing its normal strand transfer activity. Four different conformations of IN were chosen from a prior molecular dynamics (MD) simulation on the modeled IN T66I/M154I catalytic core domain as starting points for additional MD studies. The aim of this article is to understand the dynamic features that may play roles in the catalytic activity of the double mutant enzyme in the absence of any inhibitor. Moreover, we want to verify the influence of using different starting points on the MD trajectories and associated dynamical properties. By comparison of the trajectories obtained from these MD simulations we have demonstrated that the starting point does not affect the conformational space explored by this protein and that the time of the simulation is long enough to achieve convergence for this system.     

Chemical characterization (GC/MS and NMR Fingerprinting) and bioactivities of South-African Pelargonium capitatum (L.) L' Her. (Geraniaceae) essential oil

Chemical fingerprinting of commercial Pelargonium capitatum (Geraniaceae) essential oil samples of south African origin was performed by GC, GC/MS, and (13) C- and (1) H-NMR. Thirty-seven compounds were identified, among which citronellol (32.71%) and geraniol (19.58%) were the most abundant. NMR Spectra of characteristic chemicals were provided. Broad-spectrum bioactivity properties of the oil were evaluated and compared with those of commercial Thymus vulgaris essential oil with the aim to obtain a functional profile in terms of efficacy and safety. P. capitatum essential oil provides a good performance as antimicrobial, with particular efficacy against Candida albicans strains. Antifungal activity performed against dermatophyte and phytopathogen strains revealed the latter as more sensitive, while antibacterial activity was not remarkable against both Gram-positive and Gram-negative bacteria. P. capitatum oil provided a lower antioxidant activity (IC(50) ) than that expressed by thyme essential oil, both in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and β-carotene bleaching tests. Results in photochemiluminescence (PCL) assay were negligible. To test the safety aspects of P. capitatum essential oil, mutagenic and toxicity properties were assayed by Ames test, with and without metabolic activation. Possible efficacy of P. capitatum essential oil as mutagenic protective agent against NaN(3) , 2-nitrofluorene, and 2-aminoanthracene was also assayed, providing interesting and significant antigenotoxic properties.     

Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.     

Laser Speckle Imaging: A Novel Method for Detecting Dental Erosion

Erosion is a highly prevalent condition known as a non-carious lesion that causes progressive tooth wear due to chemical processes that do not involve the action of bacteria. Speckle images proved sensitive to even minimal mineral loss from the enamel. The aim of the present study was to investigate the use of laser speckle imaging analysis in the spatial domain to quantify shifts in the microstructure of the tooth surface in an erosion model. 32 fragments of the vestibular surface of bovine incisors were divided in for groups (10 min, 20 min. 30 min and 40 min of acid etching) immersed in a cola-based beverage (pH approximately 2.5) twice a day during 7 days to create an artificial erosion. By analyzing the laser speckle contrast map (LASCA) in the eroded region compared to the sound it was observed that the LASCA map shifts, proportionally to the acid each duration, by: 18%; 23%; 39% and 44% for the 10 min; 20 min; 30 min and 40 min groups, respectively. To the best of our knowledge, this is the first study to demonstrate the correlation between speckle patterns and erosion progression.     

Binding and segmentation of multiple objects through neural oscillators inhibited by contour information

 Temporal correlation of neuronal activity has been suggested as a criterion for multiple object recognition. In this work, a two-dimensional network of simplified Wilson-Cowan oscillators is used to manage the binding and segmentation problem of a visual scene according to the connectedness Gestalt criterion. Binding is achieved via original coupling terms that link excitatory units to both excitatory and inhibitory units of adjacent neurons. These local coupling terms are time independent, i.e., they do not require Hebbian learning during the simulations. Segmentation is realized by a two-layer processing of the visual image. The first layer extracts all object contours from the image by means of “retinal cells” with an “on-center” receptive field. Information on contour is used to selectively inhibit Wilson-Cowan oscillators in the second layer, thus realizing a strong separation among neurons in different objects. Accidental synchronism between oscillations in different objects is prevented with the use of a global inhibitor, i.e., a global neuron that computes the overall activity in the Wilson-Cowan network and sends back an inhibitory signal. Simulations performed in a 50×50 neural grid with 21 different visual scenes (containing up to eight objects + background) with random initial conditions demonstrate that the network can correctly segment objects in almost 100% of cases using a single set of parameters, i.e., without the need to adjust parameters from one visual scene to the next. The network is robust with reference to dynamical noise superimposed on oscillatory neurons. Moreover, the network can segment both black objects on white background and vice versa and is able to deal with the problem of “fragmentation.” The main limitation of the network is its sensitivity to static noise superimposed on the objects. Overcoming this problem requires implementation of more robust mechanisms for contour enhancement in the first layer in agreement with mechanisms actually realized in the visual cortex. Received: 25 October 2001 / Accepted: 26 February 2003 / Published online: 20 May 2003 Correspondence to: Mauro Ursino (e-mail: mursino@deis.unibo.it, Tel.: +39-051-2093008, Fax: +39-051-2093073)