Pheochromocytoma presenting as a pancreatic mass: Avoiding a dangerous pitfall in endoscopic ultrasound-guided fine needle aspiration biopsy using GATA3 immunostain

Pheochromocytomas and sympathetic paragangliomas are rare tumours arising from chromaffin cells, producing catecholamines in various amounts. Fatal hypertensive episodes may occur perioperatively, which are preventable by alpha adrenergic receptor blockers. The perioperative mortality rate of diagnosed versus undiagnosed catecholamine-producing tumours is significant, considering that only a minority of tumours develop metastasis. Herein we describe a case of a primary adrenal pheochromocytoma referred to as a pancreatic tumour, successfully diagnosed by endoscopic ultrasound-guided fine needle aspiration biopsy, with a distinct morphology (prominent nuclear anisonucleosis, intranuclear pseudoinclusions, and multinucleation) and immunohistochemical signature.     

Regionalization of axial skeleton in the lungfish Neoceratodus forsteri (Dipnoi)

Differentiation of the axial skeleton into distinct regions, once thought to be characteristic of the Tetrapoda, also occurs in the actinopterygian Danio rerio. In these taxa, the boundary between the cervical-thoracic regions correlates with Hoxc6 expression and morphological features such as position of the pectoral fin and associated nerves, and the absence of ribs. In the lungfish Neoceratodus, a member of the extant sister taxon to the Tetrapoda, the first vertebral element to chondrify is situated well posterior to the skull, developing from somites 6 and 7 (6/7) and associated with an enlarged cranial rib and nerves innervating the pectoral fin. Two vertebral elements develop later and more anteriorly, associated with somites 4/5 and 5/6. These three elements become incorporated into the occipital region of the skull during Neoceratodus ontogeny, until the cranial rib itself articulates to the rear of the skull. These features of early development indicate a regionalization of the Neoceratodus vertebral column: the cranial rib marks the boundary between the cervical and thoracic regions, the two more anterior vertebrae lacking ribs represent the cervical region, while somites 1-4 (cranial half), lacking any vertebral development, represent the occipital region. However, the cervical region of the vertebral column is effectively lost during ontogeny of Neoceratodus. A recognizable cervical region in the tetrapod vertebral column, as in zebrafish, suggests that cervical vertebrae are not incorporated into the skull but maintained as distinct elements of the column, representing an important shift in relative developmental timing and the influence of heterochrony in this region during the fish-tetrapod transition.     

Analysis of ancient sequence motifs in the H-PPase family

The unique family of membrane-bound proton-pumping inorganic pyrophosphatases, involving pyrophosphate as the alternative to ATP, was investigated by characterizing 166 members of the UniProtKB/Swiss-Prot + UniProtKB/TrEMBL databases and available completed genomes, using sequence comparisons and a hidden Markov model based upon a conserved 57-residue region in the loop between transmembrane segments 5 and 6. The hidden Markov model was also used to search the approximately one million sequences recently reported from a large-scale sequencing project of organisms in the Sargasso Sea, resulting in additional 164 partial pyrophosphatase sequences. The strongly conserved 57-residue region was found to contain two nonapeptidyl sequences, mainly consisting of the four 'very early' proteinaceous amino acid residues Gly, Ala, Val and Asp, compatible with an ancient origin of the inorganic pyrophosphatases. The nonapeptide patterns have charged amino acid residues at positions 1, 5 and 9, are apparent binding sites for the substrate and parts of the active site, and were shown to be so specific for these enzymes that they can be used for functional assignments of unannotated genomes.     

Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.     

A theoretical study on nitric oxide reductase activity in a ba(3)-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba(3)-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a(3) and is partly reduced having some nitroxyl anion character ((3)NO(-)), and it is thus activated toward the attack by the second N-O. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu(B) is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the N-O bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the N-O bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the N-O bond and form N(2)O at a feasible rate. The cleavage of the N-O bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.     

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single lambda-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.     

Mechanism for N₂O generation in bacterial nitric oxide reductase: a quantum chemical study

The catalytic mechanism of reduction of NO to N(2)O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N(2)O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.     

Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food

The aim of this study was to investigate if consumption of ordinary carbohydrate-rich food prepared in different ways has an impact on chromosome stability, i.e., on the formation of micronucleated young erythrocytes in humans. Twenty-four persons, divided into two groups, participated during 4 days in a semi-controlled food-consumption study. One group (low-heated-food-group, LowHF-group) consumed only food boiled in water (max 100 degrees C) and the other group (high-heated-food-group, HighHF-group) consumed preferentially strongly heated (fried) food. From each of the subjects, blood samples were drawn, before and after 4 days. The frequency (f) of micronucleated (MN) very young erythrocytes (transferrin-positive reticulocytes, Trf-Ret), fMNTrf-Ret, was determined, and the difference in the frequency, before and after the eating period, was calculated. The obtained mean differences for the two groups were compared. As an indicator of highly heated food the acrylamide (AA) content in part of the consumed foodstuffs was analysed by use of LC/MS-MS and the AA intake estimated. In the blood samples the hemoglobin-adduct levels from AA were analysed as a measure of the internal AA dose. The differences between the mean fMNTrf-Ret, before and after the eating period, were -0.15 per thousand for the LowHF-group and +0.17 per thousand for the HighHF-group, p<0.005 (t-test, one-tailed). The mean total AA intake in the HighHF-group during 4 days was estimated to about 3000+/-450microg per person. For the LowHF-group, the mean AA intake was low, 20+/-10microg per person. The lowest dose of AA that caused a significant increase of micronucleated erythrocytes in mice is more than a hundred times higher than the AA level in this study. Thus, it is unlikely that the exposure to AA is the major cause behind the observed difference. The answer is probably to be found in other compounds produced at the same time during heating of the food.     

A Role for Spectrin in Dynactin‐dependent Melanosome Transport in Xenopus laevis Melanophores

The bi‐directional movement of pigment granules in frog melanophores involves the microtubule‐based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin  II and myosin  V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin  II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150^glued and Arp1/centractin, co‐localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co‐immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.