Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole.

Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.

A retrothalamic system of collateral fibers from the cerebral peduncle.

Ramón y Cajal described a sizeable bundle of collateral fibers from the cerebral peduncle and called it 'faisceau de Forel'. This bundle has been consistently ignored by recent authors, possibly because of the difficulty to visualize it in transversal section. It is described here as the fasciculus prerubralis, or FPR. It originates at the level of the transition from the nucleus subthalamicus to the substantia nigra. In sagittal-stained sections of the mouse brain stained with the rapid Golgi method it can be seen following an ascending and nearly vertical course. It arches caudalwards, and appears to terminate in the rostral tegmentum. In the cat, Nauta-stained material proved that the corticofugal fibers which contribute to this system originate preponderantly, if not exclusively, in the pericruciate gyrus. Following lesions in this cortical area one could observe, in addition to the degenerating fibers of this bundle, another system of degenerating direct cortico-tegmental fibers which coursed toward the rostral tegmentum. It is not clear whether this second degenerating system resulted from the encroachment of passing fibers in the subjacent internal capsule. The tegmental prerubral collateral fibers from the cerebral peduncle were observed in mouse, rat and cat. Possibly, they could represent a general feature of the mammalian nervous system.

Uptake and clearance of diazinon in different tissues of the European eel (Anguilla anguilla L.).

Acute toxicity effects of diazinon on European eel (Anguilla anguilla) was examined using short-term exposures in static conditions. The LC50 values were 0.16, 0.11, 0.09 and 0.08 mg/l at 24, 48, 72 and 96 h exposure, respectively. In addition, the eels were exposed to 0.042 mg/l of diazinon and bioaccumulation and clearance processes of this insecticide in liver, muscle and gill tissues were studied. Bioconcentration factor (BCF) of diazinon over 96 h exposure period was 1850 in liver, 875 in gill and 775 in muscle tissue. Diazinon elimination from the selected tissues was rapid. The excretion rate constants of this insecticide were 0.108 h-1 for liver, 0.055 h-1 for gill and 0.016 h-1 for muscle. Diazinon half-lives were calculated as 16.6, 33.2 and 27 h for liver, muscle and gill, respectively. Diazinon showed relatively short half-lives in eel tissues.

Biochemical stress response in tetradifon exposed Daphnia magna and its relationship to individual growth and reproduction.

Environmental risk assessment of chemicals toxicity requires the use of costly and labor-intensive chronic data and short-term tests provide additional information. Energy budget is used by the animals for their growth, reproduction, and metabolism and it is reduced in case of toxic stress. Tetradifon acaricide is frequently used in the European Mediterranean region and it is implicated in aquatic environmental pollution. Previous studies showed that the EC(50)-24 h of tetradifon on Daphnia magna was 8.92 mg/L. Based on that, D. magna were exposed to sublethal tetradifon concentrations of 0.10, 0.18, 0.22 and 0.44 mg/L for five days in order to investigate their effect on intermediate metabolism. Caloric content was determined as biomarker of tetradifon toxicity. Results were analyzed using one-way analysis of variance (ANOVA) and Duncan's significant difference test was used to find differences between groups (alpha was set at p=0.05). Daphnids energy content decreased as tetradifon concentration increased. At 120-h caloric content was depleted >51% at pesticide concentrations of 0.18 mg/L and higher. In order to determine a possible link between the 5-d test and the 21-d chronic test, animals under short-term test were exposed to the same pesticide concentrations known to cause adverse effects on reproduction, growth and survival. Results from the present study indicated a good correlation between the proposed 5-day test and daphnid energy budget. Comparison between both, caloric content results and the chronic effect values obtained using life-table studies, suggested a good fit between them. These studies can be used as earlier, predictive and useful tests with comparable results to the classic chronic ones. Our results indicate that caution must be done about the use of tetradifon in the aquatic environment.

Non-synaptic chemical neurotransmission.

Images with apparently gemmulofugal polarity in the EPL of the olfactory bulb are the result of sectioning, along misleading planes, gemmulopetal synapses containing postsynaptic vesicles. Unless one accepts a bidirectional conduction for chemical synapses, the internal granule cells lack actual gemmulofugal synapses and the neurotransmitter contained on their vesicles must act at non-synaptic membranes.

Subcortical projections of the pericruciate cortex of cat. An autoradiography study.

This communication deals with an autoratiographic analysis of two experiments in which multiple injections of 35S methionine were performed around the cruciate sulcus of cat. One case was studied in coronal sections and the other in sagittal sections. The thalamic projections include: (1) the anterior pole of nucleus reticularis; (2) the rostral pole of the ventral thalamus (VA and VL); (3) parts of the ventrobasal complex (VPLm and (VPM); (4) intralaminary centers, in particular CL; and (5) a region around the ventral end of the fasciculus retroflexus. Instead of a terminal projection, the latter appears to receive a system of collateral fibers which leave the cerebral peduncle at meso-diencephalic levels. An intricate pattern of negatively outlined centers was found. One of them ("nucleus Q") appears as a relatively large, non-labelled field within the ventrobasal thalamus, surrounded by a "halo" of labelled territories from which it is separated by a sharp outline. Other circumscribed negatively outlined fields appear as "islands" interposed between CL and VL. Surprisingly, hardly any target structures were seen beyond the mesencephalon, with the exception of the ipsilateral pontine nuclei and the so called lateral reticular nucleus of the medulla oblongata, contralaterally. The pyramidal tract was well labelled at all levels indicating a large projection to the spinal cord. The reason why no cortico-reticular projections were labelled is not clear.

Diencephalic projections from rostral brain in cat. An autoradiographic study.

Following isotope injections in gyrus proreus (cases CT-600 and CT-601), the following nuclei were labelled: reticularis anterior, ventralis anterior, medialis dorsalis, ventralis medialis, medial portions of lateralis posterior, paratenialis and parafascicularis. Following an injection which included the medial precruciate and caudal genualis cortices (CT-625) the projection included: reticularis anterior, anterior medialis, ventralis anterior, ventralis anteromedialis, ventralis medialis, medialis dorsalis (pars paralamellaris), lateralis posterior, and parafascicularis. Thus, the medial precruciate cortex and the gyrus proreus share a number of projections. The most important differences are as follows: medialis dorsali principalis (both its medial magnocellular portion and its lateral parvicellular portion) receives afferents from gyrus proreus, whereas its paralamellar portion receives them from the medial precruciate and genualis cortices; the nucleus anterior medialis receives fibers from the medial precruciate cortex but not from gyrus proreus; a dorsolateral extension of VM, coined here as nucleus ventralis anteromedialis (VAM) receives fibers from medial precruciate but not from proreus cortex. No labelling was found in the hypothalamus, septum, and amygdala following injections in either proreus or medial precruciate cortices.

A note on the corticocaudate connexions in cat.

Following injections of labelled amino acids around the pericruciate gyrus and the medial aspect of gyrus genualis-proreus in cat, two entirely different projection patterns are found in the head of the caudate nucleus. Massive and multiple pericruciate injections failed to label the most medial caudate nucleus which, by contrast, appears selectively labelled following genualis-proreus injections. The respective accepted connexions of pericruciate and genualis-proreus cortices with the thalamus and other subcortical structures are different. Therefore, it appears that the lateral and medial regions of the caudate nucleus must differ in their hodological and, possibly, in their functional characteristics.

Pigment content of D1-D2-cytochrome b559 reaction center preparations after removal of CP47 contamination: an immunological study.

Isolated D1-D2-cytochrome b559 photosystem II reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction center prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose-12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any cross-reaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D1-D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-D1-D2-Cyt b559 complex and that native photosystem II reaction centers contain 4 core chlorophylls per 2 pheophytins.