Identification of Toxoplasma calcium-dependent protein kinase 3 as a stress-activated elongation factor 2 kinase.

Toxoplasma gondii is an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle, tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites, the elongation phase of protein synthesis is regulated by the Toxoplasma oxygen-sensing protein, PHYb. PHYb acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PHYb, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to the Saccharomyces cerevisiae eEF2 kinase, RCK2. In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions, CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular stress-induced eEF2 kinase.IMPORTANCEAlthough it is an obligate intracellular parasite, Toxoplasma must be able to survive in the extracellular environment. Our previous work indicated that ensuring that elongation continues during protein synthesis is part of this stress response and that this is due to preventing phosphorylation of elongation factor 2. But the identity of the eEF2 kinase has remained unknown in Toxoplasma and other protozoan parasites. Here, we identify CDPK3 as the first protozoan eEF2 kinase and demonstrate that it is part of a stress response initiated when parasites are exposed to extracellular stress. We also demonstrate that CDPK3 engages eEF2 as a result of its relocalization from the plasma membrane to the cytosol.

Distance of the cervical part of the internal carotid artery from the selected anatomical structures in the parapharyngeal space and its relation to patient characteristics.

Anomalies of the internal carotid artery (ICA) can increase the risk of haemorrhage during common surgical procedures. The aim of this literature review was to summarize the current state of knowledge on the course of the internal carotid artery in the parapharyngeal space, including the impact of the patient characteristics on the distances between the artery and other anatomical structures, as well as symptoms accompanying the aberrations. Pathologies related to the course of ICA in the parapharyngeal space are common (10%-60% in the general population and up to 84.4% in the elderly). In women, the distances in the oropharynx area are shorter than in men. Although the number of morphological studies is growing, providing more information on this topic, the identified studies differ in the methods and results. Knowledge on the variability in the course of ICA can help identify patients at high risk for the ICA trauma during pharyngeal procedures.

Infrared Thermography as an Indicator of Heat Loss in Fur-Chewing Chinchillas (Chinchilla Lanigera).

Fur-chewing is a common problem in chinchilla (Chinchilla lanigera). It may affect the welfare of animals due to heat loss, thereby possibly impacting food and water intake to maintain body temperature. In this context, infrared thermography seems to be a suitable method of measuring heat emissions from the surface of objects. Sexually mature male domestic chinchillas were divided into two groups: "non-fur chewers", exhibiting normal behaviour (n = 25), and "fur chewers" displaying fur-chewing behaviour (n = 23). Food and water intake (mean ± SD) measured in the control animals amounted to 20.7 ± 4.52 g and 15.9 ± 3.45 mL, while in fur-chewing chinchillas were 25% and 33% higher, respectively. Metabolic energy intake, were calculated 2.2 and 2.8 W for the control and fur-chewing animals, respectively. Heat flux through chewed areas was 6.06 mW cm-2, which is 2.8 times higher than through undamaged chinchilla fur. To sum up, thermal imagingexplicitly shows that fur-chewing causes increased heat loss. Disturbances in the maintenance of thermal homeostasis may be an additional factor that reduces the welfare of these animals.

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

The Activin Receptor, Activin-Like Kinase 4, Mediates Toxoplasma Gondii Activation of Hypoxia Inducible Factor-1.

To grow and cause disease, intracellular pathogens modulate host cell processes. Identifying these processes as well as the mechanisms used by the pathogens to manipulate them is important for the development of more effective therapeutics. As an example, the intracellular parasite Toxoplasma gondii induces a wide variety of changes to its host cell, including altered membrane trafficking, cytoskeletal reorganization, and differential gene expression. Although several parasite molecules and their host targets have been identified that mediate- these changes, few are known to be required for parasite replication. One exception is the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), which is required for parasite replication in an oxygen-dependent manner. Toxoplasma activates HIF-1 by stabilizing the HIF-1α subunit, and this is dependent on the signaling from the Activin-Like Kinase (ALK) receptor superfamily. Here, we demonstrate that specific overexpression of the ALK family member, ALK4, increased HIF-1 activity in Toxoplasma-infected cells, and this increase required ALK4 kinase activity. Moreover, Toxoplasma stimulated ALK4 to dimerize with its co-receptor, ActRII, and also increased ALK4 kinase activity, thereby demonstrating that Toxoplasma activates the ALK4 receptor. ALK4 activation of HIF-1 was independent of canonical SMAD signaling but rather was dependent on the non-canonical Rho GTPase and JNK MAP kinase signaling pathways. Finally, Toxoplasma increased rates of ALK4 ubiquitination and turnover. These data provide the first evidence indicating that ALK4 signaling is a target for a microbial pathogen to manipulate its host cell.

The anatomical relation of the extracranial internal carotid artery in the parapharyngeal space.

BACKGROUND: The proximity of the internal carotid artery to the pharyngeal wall poses a risk of injury during nasopharyngeal surgery. OBJECTIVES: The aim of this study was to assess the distances between the extracranial internal carotid artery (ICA) and the pharyngeal wall. MATERIAL AND METHODS: Measurements were taken on certain levels of the pharynx using computed tomography angiography (angio-CT) scans of 97 patients. One-tailed Student's t-test for independent variables and a comparison of expected values for dependent pairs of observations were applied. RESULTS: The shortest distance between the ICA and the pharyngeal wall was 1.1 mm. The ICA is closer to the pharyngeal wall at the epiglottis apex level (16.46 ±0.89 mm) than to the Eustachian tube (ET) (19.8 ±0.62 mm) (p < 0.0005). In women, the ICA is closer to the ET (19.44 ±0.78 mm) than in men (20.17 ±0.96 mm) (p = 0.04). In women, the right ICA is closer to the pharyngeal wall than the left ICA at the level of the lower margin of the 2nd cervical corpus vertebra (C2) (right: 17.6 ±1.8 mm; left: 20.7 ±1.7 mm) (p = 0.002) and at the level of the epiglottis apex (right: 15.2 ±1.7 mm; left: 17.4 ±1.4 mm) (p = 0.028). The bifurcation of the common carotid artery (CCA) is higher in men (19.48 ±2.19 mm below the C2) than in women (21.82 ±1.02 mm) (p < 0.001). When the bifurcation is at the level of the epiglottis apex, the ICA is closer to the pharyngeal wall (12.3 ±1.69 mm) than in other cases (16.46 ±0.89 mm) (p = 0.005). In men, the higher the bifurcation is, the closer the ICA is to the pharyngeal wall at the level of the lower margin of the C2 (p = 0.003). CONCLUSIONS: The risk of ICA incision during surgery differs between the pharyngeal levels, genders and sides of the neck. The ICA may be much closer to the pharyngeal wall than described in the literature.

Markers of anaphylaxis - a systematic review.

Anaphylaxis is defined as severe, life-threatening, systemic or general, immediate reaction of hypersensitivity, with repeatable symptoms caused by the dose of stimulus which is well tolerated by healthy persons. The proper diagnosis, immediate treatment and differential diagnosis are crucial for saving patient's life. However, anaphylaxis is relatively frequently misdiagnosed or confused with other clinical entities. Thus, there is a continuous need for identifying detectable markers improving the proper diagnosis of anaphylaxis. Here we presented currently known markers of anaphylaxis and discussed in more detail the most clinically valuable ones: tryptase, platelet activacting factor (PAF), PAF-acethylhydrolase, histamine and its metabolites.

Genomic upregulation of cardiac Cav1.2α and NCX1 by estrogen in women.

BACKGROUND: Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts. METHODS: Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs. RESULTS: In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1-2 days) increased ICa,L (31%, P < 0.05) and INCX (7.5-fold, - 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter ICa,L and INCX. CONCLUSIONS: The results show that estrogen upregulates cardiac ICa,L and INCX in women through genomic mechanisms that account for sex differences in Ca2+ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.

Biological Activities of Uric Acid in Infection Due to Enteropathogenic and Shiga-Toxigenic Escherichia coli.

In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli(EPEC) and Shiga-toxigenic E. coli(STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and release of extracellular DNA from host cells. While uric acid alone was unable to trigger a chloride secretory response in intestinal monolayers, it did potentiate the secretory response to cyclic AMP agonists. Uric acid crystals were formed in vivo in the lumen of the gut in response to EPEC and STEC infections. While trying to visualize uric acid crystals formed during EPEC and STEC infections, we noticed that uric acid crystals became enmeshed in the neutrophilic extracellular traps (NETs) produced from host cells in response to bacteria in cultured cell systems and in the intestine in vivo Uric acid levels in the gut lumen increased in response to exogenous DNA, and these increases were enhanced by the actions of DNase I. Interestingly, addition of DNase I reduced the numbers of EPEC bacteria recovered after a 20-h infection and protected against EPEC-induced histologic damage.

The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole.

Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access.

Study familial hypertrophic cardiomyopathy using patient-specific induced pluripotent stem cells.

AIMS: Familial hypertrophic cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. METHODS AND RESULTS: To investigate the molecular mechanism underlying the abnormal CM functions in HCM, we derived iPSCs from an HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for 'Cell Proliferation' was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical treatments. CONCLUSION: Overall, this study explored the possible patient-specific and mutation-specific disease mechanism of HCM, and demonstrates the potential of using HCM iPSC-CMs for future development of therapeutic strategies. Additionally, the whole methodology established in this study could be utilized to study mechanisms of other human-inherited heart diseases.

Electronic "expression" of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells.

BACKGROUND: Human-induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which human myocyte function and dysfunction are studied, especially those from patients with genetic disorders. They are also considered a major advance for drug safety testing. However, these cells have considerable unexplored potential limitations when applied to quantitative action potential (AP) analysis. One major factor is spontaneous activity and resulting variability and potentially anomalous behavior of AP parameters. OBJECTIVE: To demonstrate the effect of using an in silico interface on electronically expressed I(K1), a major component lacking in h-iPSC-derived cardiac myocytes. METHODS: An in silico interface was developed to express synthetic I(K1) in cells under whole-cell voltage clamp. RESULTS: Electronic I(K1) expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed afterdepolarizations, and decreased AP variability. The initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data obtained with freshly isolated human myocytes as well as the readily recognizable repolarization attributes of ventricular and atrial cells. The application of 1 µM of BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When I(K1) was electronically expressed, BayK-8644 prolonged the AP, which is consistent with the existing results on native cardiac myocytes. CONCLUSIONS: The electronic expression of I(K1) is a simple and robust method to significantly improve the physiological behavior of the AP and electrical profile of h-iPSC-derived cardiac myocytes. Increased stability enables the use of this preparation for a controlled quantitative analysis of AP parameters, for example, drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

Interaction of the S6 proline hinge with N-type and C-type inactivation in Kv1.4 channels.

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.

A Mouse Model of Timothy Syndrome: a Complex Autistic Disorder Resulting from a Point Mutation in Cav1.2.

Timothy Syndrome (TS) arises from a point mutation in the human voltage-gated L-type Ca2+ channel (Cav1.2). TS is associated with cardiac arrhythmias and sudden cardiac death, as well as congenital heart disease, impaired cognitive function, and autism spectrum disorders. TS results from a de novo gain-of-function mutation which affects the voltage dependent component of Cav1.2 inactivation. We created a knock-in TS mouse. No homozygous TS mice survived, but heterozygous TS2-NEO mice (with the mutation and the neocassette in situ) had a normal outward appearance and survived to reproductive age. Previously, we have demonstrated that these mice exhibit the triad of Autistic traits. In this paper we document other aspects of these mice including Cav1.2 isoform expression levels, normal physical strength, brain anatomy and a marked propensity towards self-injurious scratching. Gross brain anatomy was not markedly different in TS2-NEO mice compared to control littermates, and no missing structures were noted. The lack of obvious changes in brain structure is consistent with theTS2-NEO mice may provide a significant tool in understanding the role of calcium channel inactivation in both cardiac function and brain development.

Alternative mechanism of activation of the epithelial na+ channel by cleavage.

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of alphaENaC using the serine protease subtilisin. Trimeric channels formed with alphaFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an approximately 10-fold decrease in amiloride-sensitive whole cell conductance as compared with alphaWT (2.2 versus 21.2 microsiemens (microS)). Both alphaWT and alphaFM were activated to similar levels by subtilisin cleavage. Channels formed with alphaFD, a construct that deleted the segment between the two furin sites (Delta175-204), exhibited an intermediate conductance of 13.2 microS. More importantly, alphaFD retained the ability to be activated by subtilisin to 108.8 +/- 20.9 microS, a level not significantly different from that of subtilisin activated alphaWT (125.6 +/- 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of alpha was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with alphaLD, a construct that extended the deleted sequence of alphaFD by 17 amino acids (Delta175-221). Channels with alphaLD were uncleaved, exhibited low baseline activity (4.1 microS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.

Cholesterol induces renal vasoconstriction and anti-natriuresis by inhibiting nitric oxide production in anesthetized rats.

Although hypercholesterolemia is implicated in the pathophysiology of many renal disorders as well as hypertension, its direct actions in the kidney are not yet clearly understood. In the present study, we evaluated renal responses to administration of cholesterol (8 microg x min(-1).100 g body wt(-1); bound by polyethylene glycol) into the renal artery of anesthetized male Sprague-Dawley rats. Total renal blood flow (RBF) was measured by a Transonic flow probe, and glomerular filtration rate (GFR) was determined by Inulin clearance. In control rats (n = 8), cholesterol induced reductions of 10 +/- 2% in RBF [baseline (b) 7.6 +/- 0.3 microg x min(-1).100 g(-1)], 17 +/- 3% in urine flow (b, 10.6 +/- 0.9 microg x min(-1).100 g(-1)), 29 +/- 3% in sodium excretion (b, 0.96 +/- 0.05 mumol.min(-1).100 g(-1)) and 24 +/- 2% in nitrite/nitrate excretion (b, 0.22 +/- 0.01 nmol.min(-1).100 g(-1)) without an appreciable change in GFR (b, 0.87 +/- 0.03 ml.min(-1).100 g(-1)). These renal vasoconstrictor and anti-natriuretic responses to cholesterol were absent in rats pretreated with nitric oxide (NO) synthase inhibitor, nitro-l-arginine methylester (0.5 microg x min(-1).100 g(-1); n = 6). In rats pretreated with superoxide (O(2)(-)) scavenger tempol (50 microg x min(-1).100 g(-1); n = 6), the cholesterol-induced renal responses remained mostly unchanged, although there was a slight attenuation in anti-natriuretic response. This anti-natriuretic response to cholesterol was abolished in furosemide-pretreated rats (0.3 microg x min(-1).100 g(-1); n = 6) but remained unchanged in amiloride-pretreated rats (0.2 microg x min(-1).100 g(-1); n = 5), indicating that Na(+)/K(+)/2Cl(-) cotransport is the dominant mediator of this effect. These data demonstrate that cholesterol-induced acute renal vasoconstrictor and antinatriuretic responses are mediated by a decrease in NO production. These data also indicate that tubular effect of cholesterol on sodium reabsorption is mediated by the furosemide sensitive Na(+)/K(+)/2Cl(-) cotransporter.

Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene.

Rhodopsin kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

Hypoxia induces changes in expression of isoforms of the divalent metal transporter (DMT1) in rat pheochromocytoma (PC12) cells.

Although hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including transferrin and its receptor, little is known about the effect of low oxygen on formation of isoforms of the major iron transport protein, divalent metal transporter 1, DMT1. Accordingly, we examined the effects of hypoxia on expression and subcellular distribution of the different isoforms of DMT1 in rat PC12 cells. Treatment with low oxygen modestly increased expression of protein and mRNA levels for both the +IRE and -IRE species of DMT1. In contrast, expression of the exon 1A containing species of DMT1 was greatly increased by hypoxia as indicated by Western blot and real-time RT-PCR analysis. Message levels for the 1A isoforms increased approximately 60-fold after exposure of PC12 cells to 1% oxygen for 5 h. The subcellular distribution of exon 1A isoforms of DMT1 remained consistently in the cytoplasmic milieu of the cell after hypoxic exposure, as also did the distribution of +IRE species of DMT1. The -IRE species of DMT1, however, responded to hypoxia by becoming increasingly associated with the regions adjoining the outer cellular membranes, while a portion partially colocalized with an early endosomal marker (EEA). Hypoxia also caused a significant increase in the uptake of manganese in PC12 cells. In summary, these results demonstrate that hypoxia selectively increases expression of exon 1A containing species of DMT1 with lesser increases in either the +IRE or -IRE isoforms the transporter.

2-Methoxyestradiol inhibits proliferation of normal and neoplastic glial cells, and induces cell death, in vitro.

2-Methoxyestradiol (2ME), a metabolite of estradiol (E), inhibits proliferation of various tumor cells. In this study we determined the effect of 2ME on human glioblastoma cell lines, in vitro. We compared these cells with cultured astrocytes obtained from traumatized adult rat striatum. Exposure to 2ME had a strong antiproliferative effect on human glioblastoma and caused an increase in the population of apoptotic cells, detected by flow cytometry, in some of the investigated cell lines. A significant number of cells were blocked in the G2/M phase of the cell cycle. Concurrently, the population of cells in the G1 phase decreased in all glioblastoma cell lines. Staining with Hoechst 33258 revealed abnormal nuclear morphology in the proliferating cells treated with 2ME. Treatment with 2ME induced upregulation of wild type p53 in one of the human glioblastoma cell lines as well as in proliferating adult rat astrocytes. We conclude that 2ME inhibits the growth of human glioblastoma cell lines and induces apoptosis, in vitro. This compound deserves further investigation as a treatment for gliomas.