Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Summary Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells.Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

Mitochondrial DNA damage triggers mitochondrial dysfunction and apoptosis in oxidant-challenged lung endothelial cells

Oxidant-induced death and dysfunction of pulmonary vascular cells play important roles in the evolution of acute lung injury. In pulmonary artery endothelial cells (PAECs), oxidant-mediated damage to mitochondrial DNA (mtDNA) seems to be critical in initiating cytotoxicity inasmuch as overexpression of the mitochondrially targeted human DNA repair enzyme, human Ogg1 (hOgg1), prevents both mtDNA damage and cell death (Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL, and Gillespie MN. Am J Physiol Lung Cell Mol Physiol 283: L205-L210, 2002). The mechanism by which mtDNA damage leads to PAEC death is unknown, and the present study tested the specific hypothesis that enhanced mtDNA repair suppresses PAEC mitochondrial dysfunction and apoptosis evoked by xanthine oxidase (XO). PAECs transfected either with an adenoviral vector encoding hOgg1 linked to a mitochondrial targeting sequence or with empty vector were challenged with ascending doses of XO plus hypoxanthine. Quantitative Southern blot analyses revealed that, as expected, hOgg1 overexpression suppressed XO-induced mtDNA damage. Mitochondrial overexpression of hOgg1 also suppressed the XO-mediated loss of mitochondrial membrane potential. Importantly, hOgg1 overexpression attenuated XO-induced apoptosis as detected by suppression of caspase-3 activation, by reduced DNA fragmentation, and by a blunted appearance of condensed, fragmented nuclei. These observations suggest that mtDNA damage serves as a trigger for mitochondrial dysfunction and apoptosis in XO-treated PAECs.     

N-Acetylcysteine promotes long-term survival of cones in a model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a major source of blindness caused by a large variety of mutations that lead to the death of rod photoreceptors. After rods die, cones gradually die from progressive oxidative damage. Several types of antioxidant formulations have been shown to reduce cone cell death over a relatively short-time frame, but in order for this strategy to be translated into a new treatment for patients with RP, prolonged effects will be needed. In this study, we determined that orally administered N-acetylcysteine (NAC) reduced cone cell death and preserved cone function by reducing oxidative damage in two models of RP, rd1(+/+) and rd10(+/+) mice. In rd10(+/+) mice, supplementation of drinking water with NAC promoted partial maintenance of cone structure and function for at least 6 months. Topical application of NAC to the cornea also reduced superoxide radicals in the retina and promoted survival and functioning of cones. Since oral and/or topical administration of NAC is feasible for long-term treatment in humans, and NAC has a good safety profile, it is reasonable to consider clinical trials to evaluate the effects of prolonged treatment with NAC in patients with RP.     

Signaling pathway for nitric oxide generation with simulated ischemia in flow-adapted endothelial cells

Ischemia in the intact ventilated lung (oxygenated ischemia) leads to endothelial generation of reactive oxygen species (ROS) and nitric oxide (NO). This study investigated the signaling pathway for NO generation with oxygenated ischemia in bovine pulmonary artery endothelial cells (BPAEC) that were flow adapted in vitro. BPAECs were cultured in an artificial capillary system and subjected to abrupt cessation of flow (ischemia) under conditions where cellular oxygenation was maintained. Immunoblotting and dichlorofluorescein/triazolofluorescein fluorescence were used to assess extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and ROS/NO generation, respectively. ERK1/2 phosphorylation significantly increased during ischemia, whereas total ERK1/2 did not change. ERK1/2 phosphorylation was suppressed by an inhibitor of tyrosine phosphorylation (genestein), cholesterol-binding reagents (filipin or cyclodextrin), or inhibitors of ROS (diphenyleneiodonium, N-acetylcysteine, or catalase), suggesting a role for both membrane cholesterol and ROS in ERK1/2 activation. Ischemia resulted in a 1.8-fold increase in NO generation that was suppressed by inhibitors of ERK1/2 activation (PD-98059 or U-0126). A calmodulin inhibitor (calmidizolium) or removal of Ca2+ from the medium also blocked NO generation, indicating that endothelial NO synthase (eNOS) is the activated isoform. These results indicate ischemia induces NO generation (possibly through a membrane cholesterol-sensitive flow sensor), the ERK1/2 cascade mediates signaling from the sensor to eNOS, and ROS are required for ERK activation.     

Determination of age, growth and sexual maturity of Barbus grypus in the Dukan reservoir of Iraq

A total of 317 carp Barbus grypus were collected during one year (1977–1978) from the Dukan reservoir of Iraq to study age, grwoth and sexual maturity. The length-weight relationship was found be: log: W =–4.2298 + 2.6832 log L. and the condition ( K ) was 0.88. Back calculation was determined by using a correction factor (45.727) in Lee's equation. Predicted standard length and weight were found to be less than the actual values. The maximum increase in length was noted in the first year group, while the maximum increase in weight occurred at the time of sexual maturity. Males were found to attain maturity earlier than the females. Spawning took place during May and June.     

A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype

We have shown previously that replacement of the spike (S) gene of the apathogenic IBV strain Beau-R with that from the pathogenic strain of the same serotype, M41, resulted in an apathogenic virus, BeauR-M41(S), that conferred protection against challenge with M41. We have constructed a recombinant IBV, BeauR-4/91(S), with the genetic backbone of Beau-R but expressing the spike protein of the pathogenic IBV strain 4/91(UK), which belongs to a different serogroup as Beaudette or M41. Similar to our previous findings with BeauR-M41(S), clinical signs observations showed that the S gene of the pathogenic 4/91 virus did not confer pathogenicity to the rIBV BeauR-4/91(S). Furthermore, protection studies showed there was homologous protection; BeauR-4/91(S) conferred protection against challenge with wild type 4/91 virus as shown by the absence of clinical signs, IBV RNA assessed by qRT-PCR and the fact that no virus was isolated from tracheas removed from birds primarily infected with BeauR-4/91(S) and challenged with IBV 4/91(UK). A degree of heterologous protection against M41 challenge was observed, albeit at a lower level.Our results confirm and extend our previous findings and conclusions that swapping of the ectodomain of the S protein is a precise and effective way of generating genetically defined candidate IBV vaccines.     

Endothelial cell oxidant generation during K+-induced membrane depolarization

We tested the hypothesis that membrane depolarization may initiate oxidant generation in the endothelial cell. Depolarization was produced in bovine pulmonary arterial endothelial cells (BPAEC) in monolayer culture with varying external K⁺, or with glyburide (10 μM), tetraethylammonium (TEA, 10 mM), gramicidin (1 μM), or nigericin (2 μM). Evaluation of bisoxonol fluorescence of BPAEC indicated concentration-dependent depolarization by high K⁺ (2% change in fluorescence/mV change in membrane potential in the 5.9–48 mM range of K⁺) and essentially complete depolarization with glyburide. Generation of oxidants was assessed with o-phenylenediamine dihydrochloride (o-PD) oxidation in the presence of horseradish peroxidase (HRP). There was a time-dependent increase in o-PD oxidation with 24 mM K⁺, nigericin, and gramicidin over 2 hours compared with control. In 1 hour o-PD oxidation increased 2.8-fold for 24 mM and 3.7-fold for 48 mM K⁺ compared with control. Catalase reduced 24 mM K⁺-induced o-PD oxidation by 50%, while Cu/Zn-superoxide dismutase (SOD) abolished the increase. Oxidation of o-PD was reduced by 57% in the absence of HRP in the system. With K⁺ channel blockade, o-PD oxidation increased 3.8-fold with glyburide and 4.6-fold with TEA compared with control. These data indicate formation of H₂O₂ and possibly other oxidants with depolarization and suggest involvement of K⁺-channels in this process. © 1996 Wiley-Liss, Inc.     

Complex interactions between bovine plasminogen and streptococcal plasminogen activator PauA

The interactions between bovine plasminogen and the streptococcal plasminogen activator PauA that culminate in the generation of plasmin are not fully understood. Formation of an equimolar activation complex comprising PauA and plasminogen by non-proteolytic means is a prerequisite to the recruitment of substrate plasminogen; however the determinants that facilitate these interactions have yet to be defined. A mutagenesis strategy comprising nested deletions and random point substitutions indicated roles for both amino and carboxyl-terminal regions of PauA and identified further essential residues within the alpha domain of the plasminogen activator. A critical region within the alpha domain was identified using non-overlapping PauA peptides to block the interaction between PauA and bovine plasminogen, preventing formation of the activation complex. Homology modelling of the activation complex based upon the known structures of streptokinase complexed with human plasmin supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.     

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.