Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.     

Combining catalyzed reporter deposition-fluorescence in situ hybridization and microautoradiography to detect substrate utilization by bacteria and Archaea in the deep ocean

The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4',6'-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up l-aspartic acid (19% versus 10%).     

Contrasting Roles of Islet Resident Immunoregulatory Macrophages and Dendritic Cells in Experimental Autoimmune Type 1 Diabetes

The innate immune system critically shapes diabetogenic adaptive immunity during type 1 diabetes (T1D) pathogenesis. While the role of tissue-infiltrating monocyte-derived macrophages in T1D is well established, the role of their tissue-resident counterparts remains undefined. We now demonstrate that islet resident macrophages (IRMs) from non-autoimmune mice have an immunoregulatory phenotype and powerfully induce FoxP3+ Tregs in vitro. The immunoregulatory phenotype and function of IRMs is compromised by TLR4 activation in vitro. Moreover, as T1D approaches in NOD mice, the immunoregulatory phenotype of IRMs is diminished as is their relative abundance compared to immunostimulatory DCs. Our findings suggest that maintenance of IRM abundance and their immunoregulatory phenotype may constitute a novel therapeutic strategy to prevent and/or cure T1D.     

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor''s response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in α-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.     

Comparison of storage proteins ofPhaseolus caracalla L. with the proteins of some other representatives of the genusPhaseolus

Storage proteins of the seeds (cotyledons) of the South-American speciesPhaseolus caracalla were compared by means of immunoelectrophoretic methods with other representatives of the genusPhaseolus. These proteins most resemble the proteins of the co-called tropical group (i.e. Ph. atropurpureus, Ph. geophilus, Ph. bracteatus, Ph. semierectus) and least the so-called American endemites (Ph. vulgaris, Ph. coccineus, Ph. acutifolius, Ph. lunatus), the main globulin of which is of a completely different specificity. The proteins ofPh. caracalla are less similar to the group of the so-called Asiatic species (Ph. aureus, Ph. calcaratus, Ph. angularis, Ph. aconitifolius, Ph. trilobus) including the analyzed representatives ofVigna sinensis; their main globulin is only partly similar to that ofPh. caracalla. Some considerations on the relationship ofPh. caracalla with the so-called tropical species is presented.     

From Mesolithic hunters to Iron Age herders: a unique record of woodland use from eastern central Europe (Czech Republic)

Vegetation History and Archaeobotany - In a continuous, perfectly stratified sedimentary sequence which was discovered under a large sandstone overhang in northern Bohemia, Czech Republic, we...     

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.