Uptake In Oocytes Research Articles

SIFa and its receptors play a possibly stimulatory role during ovarian development of the mud crab Scylla paramamosain

SIFamide is an amidated neuropeptide with a conserved Ser-Ile-Phe structure at its C-terminal, which is crucial in insect courtship, sleep and feeding in insects. However, little information exists regarding its function in crustaceans. This study aimed to identify the cDNA of Sp-SIFa and its two putative receptors (Sp-SIFaR1 and Sp-SIFaR2) in the mud crab Scylla paramamosain. The expression profiles of Sp-SIFa and its receptors signaling were investigated to explore their potential roles. The results indicated that Sp-SIFa was expressed primarily in the nervous tissues and the midgut, and Sp-SIFaR1 was extensively expressed in the eyestalk ganglion, cerebral ganglion, hepatopancreas and the ovary whereas Sp-SIFaR2 was restricted to the eyestalk ganglion and ovary. The Sp-SIFa expression in the cerebral ganglion was significantly increased at the late vitellogenic stage during the ovarian development. The expression patterns of Sp-SIFaR1 and Sp-SIFaR2 in the ovary were similar to Sp-SIFa, indicating a potential function in regulating ovarian development. Subsequently, in vitro and in vivo experiments were performed to further support this hypothesis. The expression of Sp-Vg and Sp-VgR was significantly induced by Sp-SIFa peptide in vitro and in vivo. Moreover, prolonged injection of the Sp-SIFa peptide caused a significant increase in Sp-VgR expression, oocyte diameter and gonadal development index, demonstrating a possible stimulatory effect on ovarian development. In conclusion, our results suggest that Sp-SIFa may regulate ovarian development by promoting Vg biosynthesis and oocyte uptake in the mud crab S. paramamosain.

Stage-Specific L-Proline Uptake by Amino Acid Transporter Slc6a19/B0AT1 Is Required for Optimal Preimplantation Embryo Development in Mice.

L-proline (Pro) has previously been shown to support normal development of mouse embryos. Recently we have shown that Pro improves subsequent embryo development when added to fertilisation medium during in vitro fertilisation of mouse oocytes. The mechanisms by which Pro improves embryo development are still being elucidated but likely involve signalling pathways that have been observed in Pro-mediated differentiation of mouse embryonic stem cells. In this study, we show that B0AT1, a neutral amino acid transporter that accepts Pro, is expressed in mouse preimplantation embryos, along with the accessory protein ACE2. B0AT1 knockout (Slc6a19-/-) mice have decreased fertility, in terms of litter size and preimplantation embryo development in vitro. In embryos from wild-type (WT) mice, excess unlabelled Pro inhibited radiolabelled Pro uptake in oocytes and 4-8-cell stage embryos. Radiolabelled Pro uptake was reduced in 4-8-cell stage embryos, but not in oocytes, from Slc6a19-/- mice compared to those from WT mice. Other B0AT1 substrates, such as alanine and leucine, reduced uptake of Pro in WT but not in B0AT1 knockout embryos. Addition of Pro to culture medium improved embryo development. In WT embryos, Pro increased development to the cavitation stage (on day 4); whereas in B0AT1 knockout embryos Pro improved development to the 5-8-cell (day 3) and blastocyst stages (day 6) but not at cavitation (day 4), suggesting B0AT1 is the main contributor to Pro uptake on day 4 of development. Our results highlight transporter redundancy in the preimplantation embryo.

The inhibition of intestinal glucose absorption by oat‐derived avenanthramides

Avenanthramides are phenolic compounds unique to oats and may contribute to health-promoting properties associated with oat consumption. This study used Xenopus laevis oocytes expressing the glucose transporters, glucose transporter 2 (GLUT2) or sodium-glucose transport protein 1 (SGLT1) and human Caco-2 cells models to investigate the effect of oat avenanthramides on human intestinal glucose transporters. The presence of avenanthramide reduced the glucose uptake in a dose-dependent manner in Caco-2 cells. Glucose uptake in oocytes expressing either GLUT2 or SGLT1 was nullified by oat avenanthramide. There was no significant difference between the inhibition potencies of avenanthramides C and B. Thus, our results suggest that avenanthramides may contribute to the antidiabetic properties of oats. PRACTICAL APPLICATIONS: The present research focus on the antidiabetic properties of avenanthramides, which are unique phenolic compounds found in oats. Inhibiting the activities of the glucose transport proteins expressed in the small intestine is a known strategy to improve the control of postprandial glucose level. We therefore examined the inhibitory effects of avenanthramides on two glucose transporters, glucose transporter 2 and sodium-glucose transport protein 1, predominantly found in the small intestine using the human small intestinal cell model Caco-2 cell line and by heterologously expressing these two transporters in the Xenopus laevis oocytes. Based on our results, we have confirmed for the first time that the glucose uptake is indeed inhibited by the presence of avenanthramides, suggesting the possibility of incorporating avenanthramides in foods to enhance postprandial glucose response, and ultimately improve the management of diabetes. Therefore, future research could consider utilizing this evidence in the development of diabetic-friendly functional foods or nutraceuticals containing avenanthramides.

Vitellogenin receptor and fatty acid profiles of individual lipid classes of oocytes from wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle

The greater amberjack Seriola dumerili (Risso, 1810) is a large migratory pelagic fish occurring in tropical and temperate waters with a great potential for the world aquaculture industry. Previous studies showed that wild-caught female greater amberjack reared in sea cages and handled during the reproductive season, underwent extensive ovarian atresia. This atresia, however, was not related to an insufficient liver transcription or oocyte uptake of vitellogenin (Vtg). In the present study, the structure of two greater amberjack vitellogenin receptors, namely Vtgr (Lr8-) and Lrp13, was characterized. Moreover, vtgr and lrp13 gene expression and the fatty acid profiles of specific phospholipids and neutral lipids were compared in the ovaries of wild and captive-reared greater amberjack during different phases of the reproductive cycle (i.e. early gametogenesis, advanced gametogenesis and spawning). Ovarian vtgr and lrp13 transcription was more active during early gametogenesis, suggesting that vitellogenin receptor transcripts were synthesized by previtellogenic oocytes and remained in the cellular mRNA pool until oocytes resumed meiosis and entered into secondary growth (i.e. vitellogenesis). Rearing of wild-caught greater amberjack in captivity together with handling during the reproductive season was associated with a reduced vtgr and lrp13 transcription and with a diminished capacity of oocytes in the early phase of gametogenesis (primary oocyte growth) to enter into vitellogenesis. During early gametogenesis, remarkable differences in the fatty acid composition were observed between wild and captive-reared individuals: all phospholipids of captive fish displayed dramatic increases of saturates (16:0 and 18:0) and decreases of arachidonic acid (ARA) and docosahexaenoic acid (DHA). The present study confirms the susceptibility of greater amberjack reproductive function to handling stress and suggests that the consequent extensive atresia of vitellogenic follicles originated during the primary oocytes growth when the capacity of oocytes to synthesize vitellogenin receptors was reduced. The study also suggests that this reduced capacity was associated with an altered oocyte phospholipid fatty acid composition during early gametogenesis.

Methoprene-Tolerant (Met) Is Indispensable for Larval Metamorphosis and Female Reproduction in the Cotton Bollworm Helicoverpa armigera.

Juvenile hormone (JH) represses larval metamorphosis and induces adult reproduction in insects. Methoprene-tolerant (Met) is identified as an intranuclear receptor that mediates JH actions. In the present study, we characterized a Met from the severe agricultural pest, Helicoverpa armigera, namely HaMet. In the larval stage, HaMet is predominantly expressed in the epidermis and midgut, and is upregulated before each molting, whereas in adults HaMet is maximally expressed in the ovary, testis, and fat body. The immunofluorescence assay revealed that HaMet was distributed in the longitudinal and circular muscle layers of midgut in larvae, whereas in the ovary of female adults, HaMet was localized in the nucleus of the oolemma. Knockdown of HaMet in final-instar larvae shortened the time of pupation, induced abnormal pupation, and dampened pupation rate. In female adults, HaMet depletion severely suppressed the transcription of Vitellogenin (Vg) and Vitellogenin Receptor (VgR), disrupted the Vg accumulation in fat body and the yolk protein uptake in oocytes, and finally led to an impaired fecundity. Our findings therefore confirmed that HaMet acted as a nuclear receptor of JH and played an essential role in larval metamorphosis, vitellogenesis, and oocyte maturation.

How Does Oocyte Uptake Occur? A Macroscopic Study of the Ovarian and Oviductal Modifications for Egg Capture in the Coral-Snake Micrurus corallinus.

The processes of follicular development, ovulation, egg capture, and egg transport vary among vertebrates. Therefore, analysis of the reproductive tract of several lineages is needed for understanding the evolutionary changes of the reproductive system. In turtles, the ovulated eggs are released into the coelomic cavity and taken up by one of the two oviducts, a phenomenon called extrauterine migration of eggs. However, the process of egg uptake in lizards is different. The egg is ovulated directly into the infundibulum, and oocyte uptake by the contralateral oviduct rarely occurs. The same pattern has been hypothesized to occur in snakes. To test this hypothesis, we analyzed the reproductive tract of female Micrurus corallinus preserved in zoological collections. We examined the anatomical characteristics of the infundibulum and ovary and compared the reproductive output between the ovaries to verify the mechanism of egg capture and the anatomical viability of extrauterine migration of eggs. The reproductive output of the right ovary was higher than that of the left ovary, and the higher number of eggs in the right oviduct is due exclusively to the production of the ipsilateral ovary. Several anatomical features prevent extrauterine migration of eggs, including the asymmetry of the reproductive system, the arrangement of the ovarian follicles in a single row, and the formation of a wrapping around the ovary and infundibulum by the visceral pleuroperitoneum membrane (preventing against ectopic eggs). Therefore, the hypothesis of egg capture by the contralateral oviduct is anatomically infeasible in M. corallinus and possibly in other snakes. Anat Rec, 301:1936-1943, 2018. © 2018 Wiley Periodicals, Inc.

DKB114, A Mixture of Chrysanthemum Indicum Linne Flower and Cinnamomum Cassia (L.) J. Presl Bark Extracts, Improves Hyperuricemia through Inhibition of Xanthine Oxidase Activity and Increasing Urine Excretion.

Chrysanthemum indicum Linne flower (CF) and Cinnamomum cassia (L.) J. Presl bark (CB) extracts have been used as the main ingredients in several prescriptions to treat the hyperuricemia and gout in traditional medicine. In the present study, we investigated the antihyperuricemic effects of DKB114, a CF, and CB mixture, and the underlying mechanisms in vitro and in vivo. DKB114 markedly reduced serum uric acid levels in normal rats and rats with PO-induced hyperuricemia, while increasing renal uric acid excretion. Furthermore, it inhibited the activity of xanthine oxidase (XOD) in vitro and in the liver in addition to reducing hepatic uric acid production. DKB114 decreased cellular uric acid uptake in oocytes and HEK293 cells expressing human urate transporter (hURAT)1 and decreased the protein expression levels of urate transporters, URAT1, and glucose transporter, GLUT9, associated with the reabsorption of uric acid in the kidney. DKB114 exerts antihyperuricemic effects and uricosuric effects, which are accompanied, partially, by a reduction in the production of uric acid and promotion of uric acid excretion via the inhibition of XOD activity and reabsorption of uric acid. Therefore, it may have potential as a treatment for hyperuricemia and gout.

Benzylserine inhibits breast cancer cell growth by disrupting intracellular amino acid homeostasis and triggering amino acid response pathways

BackgroundCancer cells require increased levels of nutrients such as amino acids to sustain their rapid growth. In particular, leucine and glutamine have been shown to be important for growth and proliferation of some breast cancers, and therefore targeting the primary cell-surface transporters that mediate their uptake, L-type amino acid transporter 1 (LAT1) and alanine, serine, cysteine-preferring transporter 2 (ASCT2), is a potential therapeutic strategy.MethodsThe ASCT2 inhibitor, benzylserine (BenSer), is also able to block LAT1 activity, thus inhibiting both leucine and glutamine uptake. We therefore aimed to investigate the effects of BenSer in breast cancer cell lines to determine whether combined LAT1 and ASCT2 inhibition could inhibit cell growth and proliferation.ResultsBenSer treatment significantly inhibited both leucine and glutamine uptake in MCF-7, HCC1806 and MDA-MB-231 breast cancer cells, causing decreased cell viability and cell cycle progression. These effects were not primarily leucine-mediated, as BenSer was more cytostatic than the LAT family inhibitor, BCH. Oocyte uptake assays with ectopically expressed amino acid transporters identified four additional targets of BenSer, and gas chromatography-mass spectrometry (GCMS) analysis of intracellular amino acid concentrations revealed that this BenSer-mediated inhibition of amino acid uptake was sufficient to disrupt multiple pathways of amino acid metabolism, causing reduced lactate production and activation of an amino acid response (AAR) through activating transcription factor 4 (ATF4).ConclusionsTogether these data showed that BenSer blockade inhibited breast cancer cell growth and viability through disruption of intracellular amino acid homeostasis and inhibition of downstream metabolic and growth pathways.

The observed oogenesis impairment in greater amberjack Seriola dumerili (Risso, 1810) reared in captivity is not related to an insufficient liver transcription or oocyte uptake of vitellogenin

The greater amberjack Seriola dumerili is an excellent candidate for the Mediterranean aquaculture, due to its large body size and high growth rate, as well as its high flesh quality and commercial value worldwide. For its successful incorporation in the aquaculture industry, an in-depth understanding of the reproductive function of the species under rearing conditions is necessary, since completion of oogenesis in captivity is currently a bottleneck for the commercial production of the species. Liver and ovary samples from wild and captive-reared greater amberjack females were collected at three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). The cDNAs of three vitellogenins (VtgA, VtgB and VtgC) were partially sequenced and a qRT-PCR for their expression was used to compare ovarian maturity stage and liver vitellogenin transcript levels between wild and captive-reared individuals. An extensive atresia of late vitellogenic follicles, which prevented any further oocyte development and spawning was observed in captive-reared individuals during the ADVANCED phase. The expression levels of the three vitellogenins, as well as the amount of yolk globules in vitellogenic oocytes, did not differ significantly between captive-reared and wild females, indicating that the observed oogenesis impairment in greater amberjack reared in captivity was not related to an insufficient liver synthesis or a reduced oocyte uptake of vitellogenin.

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

BackgroundMonocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.Experimental approachTransport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.ResultsIn hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.ConclusionsOur results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.

Human Concentrative Nucleoside Transporter 3 Transfection with Ultrasound and Microbubbles in Nucleoside Transport Deficient HEK293 Cells Greatly Increases Gemcitabine Uptake

Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system - capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors - increased hCNT3 mRNA and 3H-gemcitabine uptake by >2,000– and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.

Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies

Human uric acid transporter 1 (hURAT1; SLC22A12) is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure–activity relationship findings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK) studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5′-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing) or intravenously (orbital sinus) administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to compute noncompartmental model PK values. Mono-oxidation (Phase I) and glucuronidation (Phase II) pathways were observed in vitro and in vivo. The in vitro data were used to compute hepatic intrinsic clearance, and the in vivo data were used to compute peak blood concentration, time after administration to achieve peak blood concentration, area under the curve, and orally absorbed fraction. The experimental data provide additional insight into the hURAT1 inhibitor structure–activity relationship and in vitro–in vivo correlation. Furthermore, the results illustrate that one may successfully prepare potent inhibitors that exhibit moderate to good oral bioavailability.

Genetic variants of organic cation transporter 1 (OCT1) and OCT2 significantly reduce lamivudine uptake

The study sought to investigate the effect of genetic variants of OCT1 (OCT1-P283L and -P341L) and OCT2 (OCT2-T199I, -T201M and -A270S), which were identified in a Korean population, on the transport of lamivudine in vitro and to compare the substrate dependent effects of OCT1 and OCT2 variants with 1-methyl-4-phenylpyridinium (MPP+), tetraethyl ammonium (TEA), metformin and lamivudine as substrates for these transporters. When the transport kinetics of lamivudine uptake in oocytes overexpressing OCT1 and OCT2 wild-type (WT) and variant proteins were measured, lamivudine uptake mediated by OCT1-WT was saturable, and uptake was decreased in oocytes expressing OCT1-P283L and -P341L variants compared with that in OCT1-WT. The Clint of lamivudine in oocytes expressing OCT1-P283L was decreased by 85.1% compared with OCT1-WT, whereas it was decreased by 48.7% in oocytes expressing OCT1-P341L. The Clint of lamivudine in oocytes expressing OCT2-T199I, -T201M and -A270S was decreased by 86.2%, 88.9% and 73.6%, respectively, compared with OCT2-WT. When comparing various substrates such as MPP+, TEA, metformin and lamivudine, the effects of the OCT1 genetic polymorphisms on their uptake were not identical. However, contrary to the case of OCT1, the uptake of MPP+, TEA, metformin and lamivudine in oocytes expressing OCT2-T199I, -T201M and -A270S variants was decreased significantly compared with that in oocytes expressing OCT2-WT. In conclusion, the effect of genetic variations of OCT1 and OCT2 on the uptake of MPP+, TEA, metformin and lamivudine was substrate-dependent.

A Conserved Aspartate Residue Located at the Extracellular End of the Binding Pocket Controls Cation Interactions in Brain Glutamate Transporters

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.

Clathrin-mediated Endocytosis: A Universe of New Questions

I'm a detail-oriented person, a watchmaker. I like to take things apart, put them back together, and understand exactly how they work. For most of the past 30 years, I have focused (some might say fixated) my attention on clathrin-coated vesicles (CCVs) and clathrin-mediated endocytosis (CME). CME, a major endocytic pathway, is involved in the ubiquitous uptake of nutrient–receptor complexes, membrane transporters, adhesion molecules, and signaling receptors, as well as in the recycling of synaptic vesicles—all of which play central roles in human health and disease. The role of coated vesicles in efficient endocytosis was first proposed in 1964 based on electron micrographs of yolk protein uptake in insect oocytes (Roth and Porter, 1964 ). CME involves the assembly of clathrin coats on the plasma membrane that concentrate receptors; invaginate; pinch off to form CCVs; and finally uncoat, releasing transport vesicles that fuse and deliver their contents to the endosomal system. To understand the mechanisms underlying CME, we and others have developed and used the now “classical” techniques of cell biology. These techniques include subcellular fractionation to purify coated vesicles (Pearse, 1975 ); biochemical fractionation coupled to proteomics to identify components of the CME machinery (Blondeau et al., 2004 ; Borner et al., 2006 ); and various cell free assays to reconstitute and mechanistically probe the uncoating reaction (Rothman and Schmid, 1986 ), CME (Smythe et al., 1992 ; Carter et al., 1993 ; Miwako and Schmid, 2005 ), and dynamin-catalyzed membrane fission (Pucadyil and Schmid, 2008 ). Consequently, during the almost 50 years since its discovery, we have learned a great deal about the molecular mechanisms underlying clathrin-mediated endocytosis (Conner and Schmid, 2003 ; Doherty and McMahon, 2009 ). Sandra L. Schmid In 1999, Jim Keen used the then newly developed green fluorescent protein (GFP) technology to tag the clathrin light chain and, for the first time, visualized CME in real time (Gaidarov et al., 1999 ). Subsequent imaging by total internal reflection fluorescence microscopy (which selectively illuminates an ∼100-nm-deep plane in the cytosol of adherent cells, thereby increasing signal-to-noise ratios) produced spectacular live-cell movies of CME. The GFP-clathrin appeared as small points of light that grew to varying intensities and then abruptly disappeared as the nascent CCVs quickly moved deeper into the cytosol and out of the zone of illumination (Merrifield et al., 2002 ). The dynamic clathrin-coated pits (CCPs) seen in these movies (see Supplemental Movie 1) are reminiscent of stars blinking in the night's sky—and they have opened up a universe of new questions that will need to be addressed over the next 50 years. Some (those of a watchmaker) reflect the need for further understanding of the molecular basis for CME, whereas others (those of a cosmologist) seek to integrate CME with the bigger picture of organismal physiology in development, health, and disease.

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K(m). We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an alpha-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60 degrees arc suggesting that TM11 is largely alpha helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.

Functional characterization of Rhesus glycoproteins from an ammoniotelic teleost, the rainbow trout, using oocyte expression and SIET analysis

Recent experimental evidence from rainbow trout suggests that gill ammonia transport may be mediated in part via Rhesus (Rh) glycoproteins. In this study we analyzed the transport properties of trout Rh proteins (Rhag, Rhbg1, Rhbg2, Rhcg1, Rhcg2, Rh30-like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [(14)C]methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with K(m) values ranging from 4.6 to 8.9 mmol l(-1). Raising external pH from 7.5 to 8.5 resulted in 3- to 4-fold elevations in J(max) values for methylamine; K(m) values were unchanged when expressed as total or protonated methylamine. Efflux of methylamine was also facilitated in Rh-expressing oocytes. Efflux and influx rates were stimulated by a pH gradient, with higher rates observed with steeper H(+) gradients. NH(4)Cl inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K(i) for ammonia against methylamine transport was 35-40% lower when expressed as total ammonia or NH(4)(+), but 5- to 6-fold higher when expressed as NH(3). With SIET we confirmed that ammonia uptake was facilitated by Rhag and Rhcg2, but not Rh30-like proteins. Ammonia uptake was saturable, with a comparable J(max) but lower K(m) value than for total or protonated methylamine. At low substrate concentrations, the ammonia uptake rate was greater than that of methylamine. The K(m) for total ammonia (560 micromol l(-1)) lies within the physiological range for trout. The results are consistent with a model whereby NH(4)(+) initially binds, but NH(3) passes through the Rh channels. We propose that Rh glycoproteins in the trout gill are low affinity, high capacity ammonia transporters that exploit the favorable pH gradient formed by the acidified gill boundary layer in order to facilitate rapid ammonia efflux when plasma ammonia concentrations are elevated.

In ovo nanoinjection of triclosan, diclofenac and carbamazepine affects embryonic development of medaka fish ( Oryzias latipes)

We examined the toxicity of three pharmaceuticals and personal care products (PPCPs) – triclosan (TCS), diclofenac (DCF), and carbamazepine (CBMZ) – on embryonic development of Japanese medaka ( Oryzias latipes) using in ovo nanoinjection. Medaka eggs (8 h post-fertilization; late blastula stage) were injected with 0.5 nL of triolein (vehicle control) or 0.5 nL of PPCPs, using different doses of TCS (1, 5, or 9 ng), DCF (1, 5, or 12 ng), or CBMZ (1, 5, or 12 ng) per egg in triolein, in addition to uninjected control. Following injection, we recorded survival, embryonic lesions, delay in embryonic development (eye, embryonic body and internal organs), heart beat rate, hatchability, and hatching time of embryos and upward swimming of larvae. As a result, injected PPCPs caused toxic responses to medaka embryos during embryonic development and around the day of hatching. Based on estimated EC 50 values of PPCPs doses on survival of injected embryos at hatching, TCS (at a dose of 4.2 ng egg –1) was generally more toxic to medaka embryos, followed by DCF (6.0 ng egg –1), and CBMZ (13.1 ng egg –1). We conclude that the nanoinjection medaka embryos model is a valuable tool for analyzing the effects of chemicals on the development of fish embryos and feasibility of nanoinjecting PPCPs into small fish eggs perhaps mimicking early exposure resulting from oocyte uptake of contaminants from maternal extra gonadal tissues.

Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300–700μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Lipoprotein mediated lipid uptake in oocytes of polychaetes (Annelida)

The uptake of the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled sex-unspecific Nereis lipoprotein was investigated in oocytes of the nereidid polychaetes Nereis virens and Platynereis dumerilii. The fluorescence label was first observed in endocytic vesicles (<1 microm diameter), which later fused to larger vesicles (2-3 microm); these were finally incorporated into existing unlabeled yolk granules (5-6 microm). In Platynereis oocytes, the fusion of endocytic vesicles was delayed in oocytes at their final stage of development compared with those at an early stage of development. Lipoprotein double-labeled with fluorescein isothiocyanate (FITC) and DiI revealed that both the protein and the lipid moiety remained co-localized during incorporation into the yolk granules of the oocyte. No labeling of the cytoplasmic lipid droplets was observed. In N. virens, unlabeled Nereis lipoprotein was effective as a competitive inhibitor of DiI-labeled Nereis lipoprotein. Ligand blot experiments demonstrated the presence of a lipoprotein receptor with an apparent molecular mass of 120 kDa, which is different from that of the known yolk protein receptor. This indicates the presence, in the polychaete oocyte, of two distinct receptors mediating yolk protein and lipoprotein uptake, respectively. Thus, the sex-unspecific lipoprotein contributes to the lipid supply of the growing oocyte in addition to the known uptake of the yolk-protein-associated lipids. The absence of label in the cytoplasmic lipid droplets, even after prolonged incubation with labeled lipoprotein, suggests that these lipids arise either by the breakdown and resynthesis of lipoprotein-derived lipids and/or by de novo synthesis within the oocyte.