Structural Basis for the Enzymatic Formation of the Key Strawberry Flavor Compound 4-Hydroxy-2,5-dimethyl-3(2H)-furanone

The last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H-dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in six different states or complexes at resolutions of ≤1.6 Å, including those with HDMF as well as three distinct substrate analogs. Our crystallographic analysis revealed a monomeric enzyme whose active site is largely determined by the bound NAD(P)H cofactor, which is embedded in a Rossmann-fold. Considering that the quasi-symmetric enolic reaction product HDMF is prone to extensive tautomerization, whereas its precursor HMMF is chemically labile in aqueous solution, we used the asymmetric and more stable surrogate product 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (EHMF) and the corresponding substrate (2E)-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone (EDHMF) to study their enzyme complexes as well. Together with deuterium-labeling experiments of EDHMF reduction by [4R-²H]NADH and chiral-phase analysis of the reaction product EHMF, our data show that the 4R-hydride of NAD(P)H is transferred to the unsaturated exocyclic C6 carbon of HMMF, resulting in a cyclic achiral enolate intermediate that subsequently becomes protonated, eventually leading to HDMF. Apart from elucidating this important reaction of the plant secondary metabolism our study provides a foundation for protein engineering of enone oxidoreductases and their application in biocatalytic processes.     

Role of nitric oxide in heparin-induced attenuation of hypoxic pulmonary vascular remodeling.

Heparin and nitric oxide (NO) attenuate changes to the pulmonary vasculature caused by prolonged hypoxia. Heparin may increase NO; therefore, we hypothesized that heparin may attenuate hypoxia-induced pulmonary vascular remodeling via a NO-mediated mechanism. In vivo, rats were exposed to normoxia (N) or hypoxia (H; 10% O(2)) with or without heparin (1,200 U x kg-1 x day-1) and/or the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg x kg-1 x day-1) for 3 days or 3 wk. Heparin attenuated increases in pulmonary arterial pressure, the percentage of muscular pulmonary vessels, and their medial thickness induced by 3 wk of H. Importantly, although L-NAME alone had no effect, it prevented these effects of heparin on vascular remodeling. In H lungs, heparin increased NOS activity and cGMP levels at 3 days and 3 wk and endothelial NOS protein expression at 3 days but not at 3 wk. In vitro, heparin (10 and 100 U x kg-1 x ml-1) increased cGMP levels after 10 min and 24 h in N and anoxic (0% O2) endothelial cell-smooth muscle cell (SMC) coculture. SMC proliferation, assessed by 5-bromo-2'-deoxyuridine incorporation during a 3-h incubation period, was decreased by heparin under N, but not anoxic, conditions. The antiproliferative effects of heparin were not altered by L-NAME. In conclusion, the in vivo results suggest that attenuation of hypoxia-induced pulmonary vascular remodeling by heparin is NO mediated. Heparin increases cGMP in vitro; however, the heparin-induced decrease in SMC proliferation in the coculture model appears to be NO independent.     

L-glucose absorption in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>) is nonmediated

We previously demonstrated in intact house sparrows substantial absorption in vivo of L-glucose, the stereoisomer of D-glucose that is assumed not to interact with the intestines D-glucose transporter. Results of some studies challenge this assumption for other species. Therefore, we tested it in vitro and in vivo, based on the principle that if absorption of a compound (L-glucose) is mediated, then absorption of its tracer will be competitively inhibited by high concentrations of either the compound itself or other compounds (e.g., D-glucose) whose absorption is mediated by the same mechanism. An alternative hypothesis that L-glucose absorption is primarily paracellular predicts that its absorption in vivo will be increased (not decreased) in the presence of D-glucose, because the permeability of this pathway is supposedly enhanced when Na⁺-coupled glucose absorption occurs. First, using intact tissue in vitro, we found that uptake of tracer-radiolabeled L-glucose was not significantly inhibited by high concentrations (100 mM) of either L-glucose or 3-O-methyl-D-glucose, a non-metabolizable but actively transported D-glucose analogue. Second, using intact house sparrows, we found that fractional absorption of the L-glucose tracer was significantly increased, not reduced, when gavaged along with 200 mM 3-O-methyl-D-glucose. This result was confirmed in another experiment where L-glucose fractional absorption was significantly higher in the presence vs. absence of food in the gut. The greater absorption was apparently not due simply to longer retention time of digesta, because no significant difference was found among retention times. Our results are consistent with the idea that L-glucose is absorbed in a non-mediated fashion, largely via the paracellular pathway in vivo.Abbreviations AUC area under the curve - 3OMD-glucose 3-O-methyl-D-glucose Communicated by I.D. Hume     

Inhibition of mucin secretion in a colonic adenocarcinoma cell line by DIDS and potassium channel blockers

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.     

Diffusion of phycobilisomes on the thylakoid membranes of the cyanobacterium Synechococcus 7942. Effects of phycobilisome size, temperature, and membrane lipid composition

A variant of fluorescence recovery after photobleaching allows us to observe the diffusion of photosynthetic complexes in cyanobacterial thylakoid membranes in vivo. The unicellular cyanobacterium Synechococcus sp. PCC7942 is a wonderful model organism for fluorescence recovery after photobleaching, because it has a favorable membrane geometry and is well characterized and transformable. In Synechococcus 7942 (as in other cyanobacteria) we find that photosystem II is immobile, but phycobilisomes diffuse rapidly on the membrane surface. The diffusion coefficient is 3 x 10(-10) cm(2) s(-1) at 30 degrees C. This shows that the association of phycobilisomes with reaction centers is dynamic; there are no stable phycobilisome-reaction center complexes in vivo. We report the effects of mutations that change the phycobilisome size and membrane lipid composition. 1) In a mutant with no phycobilisome rods, the phycobilisomes remain mobile with a slightly faster diffusion coefficient. This confirms that the diffusion we observe is of intact phycobilisomes rather than detached rod elements. The faster diffusion coefficient in the mutant indicates that the rate of diffusion is partly determined by the phycobilisome size. 2) The temperature dependence of the phycobilisome diffusion coefficient indicates that the phycobilisomes have no integral membrane domain. It is likely that association with the membrane is mediated by multiple weak interactions with lipid head groups. 3) Changing the lipid composition of the thylakoid membrane has a dramatic effect on phycobilisome mobility. The results cannot be explained in terms of changes in the fluidity of the membrane; they suggest that lipids play a role in controlling phycobilisome-reaction center interaction.     

The motion of a single molecule,the lambda-receptor,in the bacterial outer membrane

Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion constant of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial membrane.     

Solubility and posttranslational regulation of GP130/F11--a neuronal GPI-linked cell adhesion molecule enriched in the neuronal membrane skeleton.

GP130 (renamed contactin) has previously been identified by its detergent insolubility and retention with the actin-containing "membrane skeleton" isolated from chicken neurons and brain. The contactin sequence predicted a transmembrane and cytoplasmic domain for the molecule. Recently, F11 was shown to have an identical sequence except for the C terminus, and it was predicted to be linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) group. Here we describe that GP130 can be released both from brain membranes and the detergent-insoluble membrane skeleton by a phosphoinositol-specific phospholipase C (PI-PLC) indicating that F11 and GP130/contactin are probably identical and that surprisingly the lipid anchor is partly or totally responsible for its non-ionic detergent insolubility. The "membrane skeleton" is a rich source of GPI-linked glycoproteins as judged by 1) most glycoproteins can be released by a PI-PLC and 2) most [3H]ethanolamine-labeled glycoproteins are present in, or enriched in the membrane skeleton. Thus, detergent insolubility appears to be a characteristic of GPI-anchored glycoproteins. No evidence has been obtained that GP130/F11 is released or secreted in vivo or in culture. In addition, GP130/F11 has an unusually long half-life in culture of greater than 3 days. The structure of the neuronal membrane skeleton and the potential function of GPI-anchored glycoproteins is discussed.     

In vivo restitution of airway epithelium

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-m wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [³H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 m/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.     

Vasodilator effects of L-arginine are stereospecific and augmented by insulin in humans

The amino acid l-arginine, the precursor of nitric oxide (NO) synthesis, induces vasodilation in vivo, but the mechanism behind this effect is unclear. There is, however, some evidence to assume that the l-arginine membrane transport capacity is dependent on insulin plasma levels. We hypothesized that vasodilator effects of l-arginine may be dependent on insulin plasma levels. Accordingly, we performed two randomized, double-blind crossover studies in healthy male subjects. In protocol 1 (n = 15), subjects received an infusion of insulin (6 mU x kg(-1) x min(-1) for 120 min) or placebo and, during the last 30 min, l-arginine or d-arginine (1 g/min for 30 min) x In protocol 2 (n = 8), subjects received l-arginine in stepwise increasing doses in the presence (1.5 mU x kg(-1) x min(-1)) or absence of insulin. Renal plasma flow and glomerular filtration rate were assessed by the para-aminohippurate and inulin plasma clearance methods, respectively. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation, and mean flow velocity in the ophthalmic artery was measured with Doppler sonography. l-arginine, but not d-arginine, significantly increased renal and ocular hemodynamic parameters. Coinfusion of l-arginine with insulin caused a dose-dependent leftward shift of the vasodilator effect of l-arginine. This stereospecific renal and ocular vasodilator potency of l-arginine is enhanced by insulin, which may result from facilitated l-arginine membrane transport, enhanced intracellular NO formation, or increased NO bioavailability.     

Disentangling Membrane Dynamics and Cell Migration; Differential Influences of F-actin and Cell-Matrix Adhesions

Cell migration is heavily interconnected with plasma membrane protrusion and retraction (collectively termed “membrane dynamics”). This makes it difficult to distinguish regulatory mechanisms that differentially influence migration and membrane dynamics. Yet such distinctions may be valuable given evidence that cancer cell invasion in 3D may be better predicted by 2D membrane dynamics than by 2D cell migration, implying a degree of functional independence between these processes. Here, we applied multi-scale single cell imaging and a systematic statistical approach to disentangle regulatory associations underlying either migration or membrane dynamics. This revealed preferential correlations between membrane dynamics and F-actin features, contrasting with an enrichment of links between cell migration and adhesion complex properties. These correlative linkages were often non-linear and therefore context-dependent, strengthening or weakening with spontaneous heterogeneity in cell behavior. More broadly, we observed that slow moving cells tend to increase in area, while fast moving cells tend to shrink, and that the size of dynamic membrane domains is independent of cell area. Overall, we define macromolecular features preferentially associated with either cell migration or membrane dynamics, enabling more specific interrogation and targeting of these processes in future.     

Effect of procaine hydrochloride on the electrophoretic mobility of human red blood cells

The interaction of cationic anesthetics with biological membranes and the resulting alterations of membrane electrokinetic properties continue to be of current interest. The present study was designed to examine the effects of procaine hydrochloride (PRHCL) on the mobility of human red blood cells (RBC); electrophoretic measurements were made on RBC suspended in phosphate-buffered saline (PBS, pH = 5.0, 7.4, or 9.2), autologous plasma or 3 g% dextran T70/PBS (pH = 7.4), with PRHCL concentrations from 8 x 10(-6) to 8 x 10(-2) M. Low concentrations of PRHCL (8 x 10(-5)-8 x 10(-3) M) significantly (p less than 0.001) increased RBC mobility, with a maximal increase of 8.2% at 8 x 10(-4) M. Conversely, a higher PRHCL concentration (8 x 10(-2) M significantly (p less than 0.001) decreased RBC mobility. Both glutaraldehyde fixation and lipid extraction abolished any PRHCL-induced increase in RBC mobility; the observed increases in mobility for normal cells are, thus, consistent with a mechanism based on expansion of the RBC membrane glycocalyx. Microelectrophoretic methods were also used to study the effect of PRHCL (8 x 10(-4) and 8 x 10(-2) M) on RBC membrane calcium binding, with the results indicating that PRHCL competes with calcium for neuraminate binding sites. We conclude that the observed changes in RBC electrokinetic properties reflect incorporation of PRHCL into the RBC membrane; such changes may be of importance in modulating cell-cell interactions.     

Concordant induction of rapid in vivo pulsatile insulin secretion by recurrent punctuated glucose infusions

Insulin is largely secreted as serial secretory bursts superimposed on basal release, insulin secretion is regulated through changes of pulse mass and frequency, and the insulin release pattern affects insulin action. Coordinate insulin release is preserved in the isolated perfused pancreas, suggesting intrapancreatic coordination. However, occurrence of glucose concentration oscillations may influence the process in vivo, as it does for ultradian oscillations. To determine if rapid pulsatile insulin release may be induced by minimal glucose infusions and to define the necessary glucose quantity, we studied six healthy individuals during brief repetitive glucose infusions of 6 and 2 mg x kg(-1) x min(-1) for 1 min every 10 min. The higher dose completely synchronized pulsatile insulin release at modest plasma glucose changes ( approximately 0.3 mM = approximately 5%), with large ( approximately 100%) amplitude insulin pulses at every single glucose induction (n = 54) at a lag time of 2 min (P < 0.05), compared with small (10%) and rare (n = 3) uninduced insulin excursions. The smaller glucose dose induced insulin pulses at lower significance levels and with considerable breakthrough insulin release. Periodicity shift from either 7- to 12-min or from 12- to 7-min intervals between consecutive glucose (6 mg x kg(-1) x min(-1)) infusions in six volunteers revealed rapid frequency changes. The orderliness of insulin release as estimated by approximate entropy (1.459 +/- 0.009 vs. 1.549 +/- 0.027, P = 0.016) was significantly improved by glucose pulse induction (n = 6; 6 mg x kg(-1) x min(-1)) compared with unstimulated insulin profiles (n = 7). We conclude that rapid in vivo oscillations in glucose may be an important regulator of pulsatile insulin secretion in humans and that the use of an intermittent pulsed glucose induction to evoke defined and recurrent insulin secretory signals may be a useful tool to unveil more subtle defects in beta-cell glucose sensitivity.     

Regulator of complement activation (RCA) gene cluster in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3′- or 5′-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr ∼67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.     

Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli

Background

The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically.

Methodology/Principal Findings

We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization.

Conclusions

Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation.

Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.     

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.     

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells

Background

Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells.

Methods

HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo.

Results

Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG.

Conclusions

miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor.

General significance

This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.     

Mechanism of in vivo DNA transport into cells by electroporation: electrophoresis across the plasma membrane may not be involved

BACKGROUND: Recently, in vivo gene transfer with electroporation (electro-gene transfer) has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines. The widely hypothesized mechanism is that electroporation induces structural defects in the membrane and provides an electrophoretic force to facilitate DNA crossing the permeabilized membrane. In this study, we have designed a device and experiments to test the hypothesis. METHODS: In this study, we have designed a device that alternates the polarity of the applied electric field to elucidate the mechanism of in vivo electro-gene transfer. We also designed experiments to challenge the theory that the low-voltage (LV) pulses cannot permeabilize the membrane and are only involved in DNA electrophoresis, and answer the arguments that (1) the reversed polarity pulses can cause opposing sides of the cell membrane to become permeabilized and provide the electrophoresis for DNA entry; or (2) once DNA enters cytoplasmic/endosomal compartments after electroporation, it may bind to cellular entities and might not be reversibly extracted. Thus a gradual buildup of the DNA in the cell still seems quite possible even under the condition of the rapid reversal of polarity. RESULTS: Our results indicate that electrophoresis does not play an important role in in vivo electro-gene transfer. CONCLUSIONS: This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.