High prevalence of buccal ulcerations in largemouth bass, Micropterus salmoides (Centrarchidae) from Michigan inland lakes associated with Myzobdella lugubris Leidy 1851 (Annelida: Hirudinea)

Widespread mouth ulcerations were observed in largemouth bass collected from eight inland lakes in the Lower Peninsula of Michigan during the summer months of 2002 and 2003. These ulcerations were associated with, and most likely caused by, leech parasitism. Through the use of morphological dichotomous keys, it was determined that all leeches collected are of one species: Myzobdella lugubris. Among the eight lakes examined, Lake Orion and Devils Lake had the highest prevalence of leech parasitism (34% and 29%, respectively) and mouth ulcerations (53% and 68%, respectively). Statistical analyses demonstrated that leech and ulcer prevalence varied significantly from one lake to the other. Additionally, it was determined that the relationship between the prevalence of ulcers and the prevalence of leech attachment is significant, indicating that leech parasitism is most likely the cause of ulceration. The ulcers exhibited deep hemorrhagic centers and raised irregular edges. Affected areas lost their epithelial lining and submucosa, with masses of bacteria colonizing the damaged tissues. Since largemouth bass is a popular global sportfish and critical to the food web of inland lakes, there are concerns that the presence of leeches, damaged buccal mucosa, and general unsightliness may negatively affect this important sportfishery.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

The adenovirus type 12 - mouse cell system: permissivity and analysis of integration patterns of viral DNA in tumor cells.

The integration patterns of persisting adenovirus type 12 (Ad12) DNA were analyzed in two Ad12-induced tumors of Balb/c and CBA/J mice and in one tumor cell line derived from an Ad12-induced retinoblastoma of C3H origin. In all three tumors the Ad12 genome was integrated colinearly and various copy numbers of viral DNA were found. Analysis of the Ad12 integration patterns revealed relatively simple offsize band patterns regardless of Ad12 copy numbers. The degree of methylation at the 5''-CCGG-3'' sites in the inserted Ad12 genome was determined using the isoschizomeric restriction endonuclease pair HpaII and MspI. Methylation was rather incomplete in the primary tumor tissues but almost complete in the retinoblastoma line carried in culture for many passages. The levels of expression of the viral genome in the Balb/c tumor and in the retinoblastoma line were determined by in vitro translation of RNA isolated from these cells and selected with appropriate restriction endonuclease fragments of Ad12 DNA. In both instances the 59 K, 19 K, and 17 K proteins of the E1b region were expressed. Proteins of the E1a region appeared very faint in the size class between 22 K and 42 K. The permissivity of Ad12 and the replication of Ad12 DNA in mouse cells were investigated by blotting restricted DNA from cells soon after, and a long time after, infection and by hybridization with 32P-labeled Ad12 DNA. Neither primary mouse kidney cells nor the established L929 mouse cell line supported viral DNA replication. These results raise the question to what extent host cell factors determine Ad12 DNA replication in mammalian cells.     

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.     

Nitrous oxide and methane fluxes of a pristine slope mire in the German National Park Harz Mountains

Pristine peatlands covered by Histosols (bogs and fens) with high water table and a restricted oxygen (O₂) availability are known to have low emissions of nitrous oxide (N₂O) but may be a significant source for atmospheric methane (CH₄) which are both important greenhouse gases. For the first time N₂O and CH₄ fluxes of a pristine slope mire in the German Harz Mountains have been monitored. Previously reported peatlands are characterised by anaerobic conditions due to high water table levels. Slope mires monitored here receive O₂ through slope water inflow. Gas fluxes have been monitored deploying closed chamber method on a central non-forested area and a forested area at the periphery of the slope mire. By means of groundwater piezometers water table levels, ammonium and nitrate contents as well as hydro-chemical variables like oxygen content and redox potential of the mire pore water have been concurrently measured with trace gas fluxes at both monitoring sites of the slope mire. The slope mire took up small amounts of atmospheric methane at a rate of −0.02 ± 0.01 kg C ha⁻¹ year⁻¹ revealing no significant difference between the forested and non-forested site. Higher uptake rates were observed during low water table level. In contrast to pristine peatlands influx of oxygen containing pore water into slope mire does limit reduction processes and resultant CH₄ emission. N₂O fluxes of the forested and non-forested sites of the slope mire did not differ and amounted to 0.25 ± 0.44 kg N ha⁻¹ year⁻¹. Higher emissions were observed at low water table levels and during thawing periods. In spite of favourable conditions N₂O fluxes of the slope mire have been comparable to those of pristine peatlands.     

Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies

Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas < or = 20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard control.     

Induction of Nitric Oxide Synthase and Nitric Oxide-Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor-α/Lipopolysaccharide

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N -monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4 H -1,3-thiazine HCl prevented TNF-α/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-α/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of l -arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-α/LPS-induced loss of viability.     

Early and late postnatal myocardial and vascular changes in a protein restriction rat model of intrauterine growth restriction

Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-β and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life.