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.     

Leucocytosis in Biomphalaria glabrata sensitized and resensitized to Echinostoma lindoense

Leucocytosis was shown to occur in the pulmonate gastropod Biomphalaria glabrata exposed to the trematode Echinostoma lindoense. In these sensitized snails, the leukocyte count in the hemolymph was elevated 3 to 5 days postexposure to miracidia, and prior to complete encapsulation of sporocysts. This increase continued 1 to 5 days after destruction of sensitizing, irradiated E. lindoense sporocysts. Counts returned to normal levels after this period. A significant and more rapid increase in numbers of circulating leukocytes occurred 1 to 6 hr after reexposure of snails to a sensitizing dose of nonirradiated E. lindoense sporocysts. The leukocyte counts usually returned to normal levels after this period, except in snails in which some resensitizing sporocysts remained alive.     

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.     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.     

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.     

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.     

Cryopreservation of <Emphasis Type="Italic">Panax ginseng</Emphasis> Adventitious Roots

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L⁻¹ was recorded for post-freeze-regenerated cultures versus 9.1 g dw L⁻¹ for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.