The binding of rat uterine cytosol oestrogen receptors to oligodeoxythymidylate--cellulose. Its relationship to a stable form of receptor complex with separate ligand- and oligonucleotide-binding sites.

The interaction of rat uterine cytosol oestrogen-receptor complexes with the synthetic acceptor oligo(dT)--cellulose was studied. Differences in the stability of receptor complexes and their ability to bind to oligo(dT)--cellulose on storage at 4 degrees C or when exposed to increased temperatures indicated heterogeneity of steroid- and oligonucleotide-binding sites. Dilution, dialysis and (NH4)2SO4 precipitation increased the interaction of receptor complexes with oligo(dT)--cellulose (a step termed activation). This increase may be the result of the removal of low-molecular-weight cytosol components which inhibit receptor activation, dimerization to the 5 S form, which binds to oligo(dT)--cellulose, or interaction of 5 S receptor with the oligonucleotide. Cytosol oestradiol--receptor complexes exhibited biphasic dissociation kinetics. All these manipulations resulted in an increase in the proportion of the slow-dissociating component equivalent to the increase in receptor binding to oligo(dT)--cellulose. In contrast, addition of 10mM-sodium molybdate to cytosol decreased both oligo(dT)--cellulose binding and the proportion of receptor with slow dissociation kinetics. The inclusion of proteinase inhibitors did not affect interactions of receptor with oligo(dT)--cellulose nor the dissociation kinetics. These results suggest that oligo(dT)--cellulose binding may serve to quantify the proportion of cytosol receptor in an active form capable of nuclear interaction and to help to ascertain whether a receptor system is fully functional. This binding procedure could prove useful in the evaluation of oestrogen responsivity under normal and pathological conditions.     

Studies on the Malayan forest rat filaria, Breinlia booliati: periodicity and microfilaraemic patterns during the course of infection.

Breinlia booliati exhibited nocturnal subperiodicity in its natural host, Rattus sabanus in contrast to experimentally infected laboratory-reared albine rats which showed irregular fluctuations of microfilariae throughout the 24 hour cycle. All the infected albino rats showed a prepatent period between 11-14 weeks postinoculation. Three patterns of microfilaraemia were discerned during the course of infection 38/49 rats displayed a single peak, 4/49 displayed 2 peaks about 12-15 weeks apart and 7/49 showed a sustained high plateau-like pattern of microfilaraemia. Cortisone had no effect on microfilarial levels when administered to rats near postpatency and some at postpatency.     

Re-Identification on Korean Penicillium Sequences in GenBank Collected by Software GenMine

Penicillium species have been actively studied in various fields, and many new and unrecorded species continue to be reported in Korea. Moreover, unidentified and misidentified Korean Penicillium species still exist in GenBank. Therefore, it is necessary to revise the Korean Penicillium inventory based on accurate identification. We collected Korean Penicillium nucleotide sequence records from GenBank using the newly developed software, GenMine, and re-identified Korean Penicillium based on the maximum likelihood trees. A total of 1681 Korean Penicillium GenBank nucleotide sequence records were collected from GenBank. In these records, 1208 strains with four major genes (Internal Transcribed Spacer rDNA region, β-tubulin, Calmodulin and RNA polymerase II) were selected for Penicillium re-identification. Among 1208 strains, 927 were identified, 82 were identified as other genera, the rest remained undetermined due to low phylogenetic resolution. Identified strains consisted of 206 Penicillium species, including 156 recorded species and 50 new species candidates. However, 37 species recorded in the national list of species in Korea were not found in GenBank. Further studies on the presence or absence of these species are required through literature investigation, additional sampling, and sequencing. Our study can be the basis for updating the Korean Penicillium inventory.     

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish

The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus. An immersion challenge model was utilized to mimic natural infection conditions in fish. Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E. tarda (PPD130/91) by histological and infection kinetics studies. On the other hand, avirulent E. tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days.     

Optimization of culture medium for cloned bovine embryos and its influence on pregnancy and delivery outcome

This study was conducted to establish an effective culture system for supporting in vitro development of cloned bovine embryos and to evaluate whether improved development in the optimal culture system could contribute to enhancing pregnancy and delivery outcomes after transfer. Enucleated oocytes at the metaphase II stage were reconstructed with serum-starved ear fibroblasts and cloned embryos were subsequently cultured for 168 h in vitro. In Experiment 1, cloned embryos were cultured in either modified Charles Rosenkrans 2 amino acid medium (mCR2aa) or modified synthetic oviduct fluid medium (mSOF). More (P < 0.05) 2-cell embryos (78% versus 92%), morulae (51% versus 69%) and blastocysts (2% versus 39%) were obtained after culture in mSOF than after culture in mCR2aa. In Experiment 2, cloned embryos were successively cultured in mSOF supplemented with various macromolecules during different periods of culture. A successive culture of oocytes in BSA-containing medium for 72 h and then in FBS-containing medium for the next 96 h yielded a higher rate of blastocyst formation (49% versus 25-36%) than other combinations (BSA to BSA or PVA to PVA, BSA or FBS). This macromolecule supplementation also significantly increased the number of total blastomeres (117.3 cells/blastocyst) and inner cell mass cells (ICM, 49.7 cells/blastocyst), and the ratio of ICM cells to trophoblast cells (TB, 0.98). In Experiment 3, a total of 85 blastocysts obtained from each 2-step culture were transferred individually to recipient cows at the end of the culture period and 32 pregnancies (38%) were diagnosed on Day 60 after transfer. However, no (P > 0.05) significant differences due to culture were apparent in the pregnancy outcome. Although six calves were produced using the 2-step culture regime of either BSA-BSA or PVA-FBS, no calves were produced using the successive culture of BSA then FBS, which optimized preimplantation development. In conclusion, mSOF has more potential to support the development of clone embryos than mCR2aa, and successive supplementation of BSA and FBS to mSOF further promotes blastocyst formation. However, enhanced development in vitro might not directly contribute to improving pregnancy outcomes.     

Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems

Maize and Arabidopsis root apical meristems differ in several aspects of their radial organization and ontogeny. Despite the large evolutionary distance and differences in root radial patterning, analysis of the putative maize ortholog of the Arabidopsis patterning gene SCARECROW (SCR) revealed expression localized to the endodermis, which is similar to its expression in Arabidopsis. Expression in maize extends through the quiescent center, a population of mitotically inactive cells formerly thought to be undifferentiated and to lack radial pattern information. Zea mays SCARECROW (ZmSCR), the putative maize SCR ortholog, was used as a molecular marker to investigate radial patterning during regeneration of the root tip after either whole or partial excision. Analysis of the dynamic expression pattern of ZmSCR as well as other markers indicates the involvement of positional information as a primary determinant in regeneration of the root radial pattern.     

Draft Genome Sequence of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T

Fusobacterium nucleatum, one of the major causative bacteria of periodontitis, is classified into five subspecies (nucleatum, polymorphum, vincentii, animalis, and fusiforme) on the basis of the several phenotypic characteristics and DNA homology. This is the first report of the draft genome sequence of F. nucleatum subsp. fusiforme ATCC 51190(T).     

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.