Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum

High-resolution flow cytometry, using avian erythrocytes as an internal standard, was employed to study constitutive genome size variation of G2-phase nuclei of Physarum polycephalum strains during the macroplasmodial stage of their life cycle. Our results document a previously unknown extent of genome size variation and mixoploidy in this organism. The unimodal diploid strain Tu 291 displayed the largest genome of the strains tested; in contrast, the Colonia strain displayed only half of the Tu 291 G2-phase fluorescence, confirming its haploid nature. An additional strain, derived from a recent cross between Lu897 and Lu898 amoebae, must have arisen by selfing (propagation of only one of the parental genomes to the macroplasmodial stage), since its nuclei display close to the haploid G2-phase DNA content. The observation of a small fraction of corresponding diploid nuclei within the haploid population of this strain, while maintained as microplasmodia, supports the notion that meiosis in haploid strains may require the presence of diploid nuclei. Two of the descendants of the prototype haploid Colonia strain, which were kept for extended periods of time in submerse culture, proved to be near diploid and mixoploid. Polyploidization and subsequent loss of DNA thus seems to contribute to the extremes of genome size variation in Physarum. In addition to unimodal fluorescence distributions, a number of diploid strains displayed bi- and even trimodal distributions within harvests of a single G2-phase macroplasmodium. Analysis of these mixoploid strains by means of gaussian curve-fitting suggests that the smaller genome size differences in Physarum may arise in step-wise diminution of DNA in approximate units of 3-5% of the original Tu 291 genome.     

Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase

On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca²⁺-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker -mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca²⁺-ATPase, sarco(endo)plasmic reticulum Ca²⁺-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La³⁺ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells. calcium transport     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

The lateral line receptor array of cyprinids from different habitats

The lateral line system of teleost fishes consists of an array of superficial and canal neuromasts (CN). Number and distribution of neuromasts and the morphology of the lateral line canals vary across species. We investigated the morphology of the lateral line system in four diurnal European cyprinids, the limnophilic bitterling (Rhodeus sericeus), the indifferent gudgeon (Gobio gobio), and ide (Leuciscus idus), and the rheophilic minnow (Phoxinus phoxinus). All fish had lateral line canals on head and trunk. The total number of both, CN and superficial neuromasts (SN), was comparable in minnow and ide but was greater than in gudgeon and bitterling. The ratio of SNs to CNs for the head was comparable in minnow and bitterling but was greater in gudgeon and ide. The SN‐to‐CN ratio for the trunk was greatest in bitterling. Polarization of hair cells in CNs was in the direction of the canal. Polarization of hair cells in SNs depended on body area. In cephalic SNs, hair cell polarization was dorso‐ventral or rostro‐caudal. In trunk SNs, it was rostro‐caudal on lateral line scales and dorso‐ventral on other trunk scales. On the caudal fin, hair cell polarization was rostro‐caudal. The data show that, in the four species studied here, number, distribution, and orientation of CNs and SNs cannot be unequivocally related to habitat. J. Morphol. 275:357–370, 2014. © 2013 Wiley Periodicals, Inc.     

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

Introduction

Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.

Methods

We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.

Results

Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻⁷ M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.

Conclusions

We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.     

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.     

Geographic variations in underwater male Weddell seal Trills suggest breeding area fidelity

Adult Weddell seals (Leptonychotes weddellii) exhibit site fidelity to where they first breed but juveniles, and perhaps transient adult males, may disperse from their natal location. If there is mixing between adjacent breeding groups, we would expect that common vocalizations would exhibit clinal patterns. Underwater Trill vocalizations of male Weddell seals at Mawson, Davis, Casey, McMurdo Sound, Neumayer and Drescher Inlet separated by ca. 500 to >9,000 km, were examined for evidence of clinal variation. Trills are only emitted by males and have a known territorial defense function. Trills from Davis and Mawson, ca. 630 km apart, were distinct from each other and exhibited the greatest number of unique frequency contour patterns. The acoustic features (duration, waveform, frequency contour) of Trills from Neumayer and Drescher Inlet, ca. 500 km apart, were more distinct from each other than they were from the other four locations. General Discriminant Analysis and Classification Tree Analysis correctly classified 65.8 and 76.9% of the Trills to the correct location. The classification errors assigned more locations to sites >630 km away than to nearest neighbours. Weddell seal Trills exhibit geographic variation but there is no evidence of a clinal pattern. This suggests that males remain close to single breeding areas throughout their lifetime.     

Immunocytochemical and electron-microscopic investigations of the pineal organ in adult agamid lizards,Uromastix hardwicki

Summary Lacertilian species display a remarkable diversity in the organization of the neural apparatus of their pineal organ (epiphysis cerebri). The occurrence of immunoreactive S-antigen and opsin was investigated in the retina and pineal organ of adult lizards, Uromastix hardwicki. In this species, numerous retinal photoreceptors displayed S-antigen-like immunoreactivity, whereas only very few pinealocytes were labeled. Immunoreactive opsin was found neither in retinal photoreceptors nor in pinealocytes. Electron microscopy showed that all pinealocytes of Uromastix hardwicki resemble modified pineal photoreceptors. A peculiar observation is the existence of a previously undescribed membrane system in the inner segments of these cells. It is evidently derived from the rough endoplasmic reticulum but consists of smooth membranes. The modified pineal photoreceptor cells of Uromastix hardwicki were never seen to establish synaptic contacts with somata or dendrites of intrapineal neurons, which are extremely rare. Vesiclecrowned ribbons are prominent in the basal processes of the receptor cells, facing the basal lamina or establishing receptor-receptor and receptor-interstitial type synaptoid contacts. Dense-core granules (60–250 nm in diameter) speak in favor of a secretory activity of the pinealocytes. Attention is drawn to the existence of receptor-receptor and receptor-interstitial cell contacts indicating intramural cellular relationships that deserve further study.Supported by the Deutsche Forschungsgemeinschaft (Ko 758/31) and the Deutscher Akademischer Austauschdienst (Senior DAAD Research Fellowship to M.A.H.)