Vectorial Transfer Research Articles

A 3D vectorial optical transfer function suitable for arbitrary pupil functions

We calculate the 3D vectorial optical transfer function directly from the vectorial pupil function, without making the paraxial assumption nor assuming radially symmetric pupils. Our model uses a single autocorrelation integral with Cartesian pupil co-ordinates to calculate the transfer function. Results for Herschel and aplanatic systems are presented. We discuss the meaning and application of a vectorial transfer function as a tool for analysing high aperture incoherent microscopy modes including fluorescence and transmission.

Cover Picture: Angew. Chem. Int. Ed. 11/2002

The cover picture shows the calculated energy-minimized 3D-structure of a polyphenylene dendrimer, which was prepared in a multistep synthesis. The nanoparticle carries three different types of chromophores, located in the center (terrylene), within the scaffold (perylene), and at the periphery (naphthalene). This multichromophoric triad absorbs over the whole range of the visible spectrum and shows, as shown in the picture, a stepwise vectorial energy transfer over a distance of 30 A from the surface to the core. There is more about polyphenylene chromophores in the communication from Mullen et al. on p. 1900 ff.

Fusion of chromatophores from photosynthetic bacteria with a supported lipid layer: characterization of the electric units

Direct electrometric measurements of membrane potential changes are a valuable tool for study of vectorial transfer of electrons, protons, and ions. Commonly model membrane systems are created by fusion of lipid/protein vesicles with lipid-coated thin films. We characterized the electric units resulting from this process using chromatophores from the purple bacterium Rhodobacter sphaeroides and either a Mylar film or a planar modified gold electrode as support. Investigation of the shunting activity of the ionophore gramicidin on the flash-induced potential change demonstrates fusion of individual chromatophores to form independent ‘blisters’, which preserve an interior aqueous compartment. Under current–clamp conditions the photovoltage follows the change of the membrane potential of the individual blisters.

Expression and Differential Polarization of the Reduced-folate Transporter-1 and the Folate Receptor α in Mammalian Retinal Pigment Epithelium

The differential polarized distribution of the reduced- folate transporter (RFT-1) and folate receptor alpha (FRalpha), the two proteins involved in the transport of folate, has been characterized in normal mouse retinal pigment epithelium (RPE) and in cultured human RPE cells. RPE cells mediate the vectorial transfer of nutrients from choroidal blood to neural retina. Whereas FRalpha is known to be present in many cell types of the neural retina, in situ hybridization analysis in the present study demonstrated that RFT-1 is present only in RPE. Laser-scanning confocal microscopy using antibodies specific for RFT-1 demonstrated an apical distribution of this protein in cultured human and intact mouse RPE, which contrasts with the basolateral distribution of FRalpha in these cells. The expression of RFT-1 in the RPE cell apical membrane was confirmed by functional studies with purified apical membrane vesicles from bovine RPE. These studies, done with N(5)-methyltetrahydrofolate (the predominant folate derivative in blood) and folate as substrates, have shown that RFT-1 functions in a Na(+)- and C1(-)-independent manner. The transporter is specific for folate and its analogs. A transmembrane H(+) gradient influences the transport function of this protein markedly; the transport mechanism is likely to be either folate/H(+) co-transport or folate/OH(-) exchange. Based on the differential polarization of FRalpha and RFT-1 in RPE, we suggest that these two proteins work in a concerted manner to bring about the vectorial transfer of folate across the RPE cell layer from the choroidal blood to the neural retina. This constitutes the first report of the differential polarization of the two folate transport proteins in any polarized epithelium.

Photoinduced vectorial charge transfer in mixed Langmuir–Blodgett films of 5,10,15,20-tetrakis(3,5-di-tert-butylphenyl)porphyrin and its zinc(ii) derivative

Langmuir–Blodgett films containing 1 mol% 5,10,15,20-tetrakis(3,5-di-tert-butylphenyl)porphyrin (TBP) and its zinc(II) complex (ZnTBP) in a suitable matrix were prepared in an attempt to build a molecular-scale device capable of vectorial photoinduced electron transfer. The films were studied by absorption and fluorescence spectroscopies, and by the time-resolved measurement of Maxwell displacement charge (TRMDC) across the film. The latter experiments showed that photoinduced electron transfer takes place in the films and that its macroscopic direction can be controlled by the molecular orientation. The TRMDC signals were unusually long-lived.

Polarized Distribution of Na+-Dependent Glucose Cotransporter SGLT1 in Epithelial Cells.

SGLT1, an isoform of Na+−dependent glucose cotransporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney, where it plays a pivotal role in the absorption and reabsorption of sugars, respectively. Preferential localization of SGLT1 in the apical plasma membrane domain of these epithelial cells is crucial in the vectorial transfer of sugars. Mutations of SGLT1 gene have been found to be responsible for the congenital glucose galactose malabsorption syndrome. These mutant genes, when expressed in Xenopus oocytes, resulted in the alteration in their cellular localization. In this review, we focus on the molecular mechanism for the localization of SGLT1 in the polarized epithelial cells.

Vectorial diffraction patterns and limits of Brewster ellipsometry

A vectorial modulation transfer function approach of diffraction patterns observable around Brewster's incidence is proposed. The good agreement between experimental results and vectorial model will allow the correlation of refractive index measurement accuracy with the average magnitude of surface defects. Consequences in the metrology of refractive index gradients will then be briefly discussed.

Imaging with polarized light: the vectorial modulation transfer function of a polarizing plate

A vectorial modulation transfer function or spatial frequency response of optical imaging systems including linearly polarizing components is defined, calculated and compared to experimental results and to the classical scalar paraxial theory. This exact linear polarizer modulation transfer function calculus could be of particular interest in new ellipsometric measurement methods accuracy, especially for high resolution metrology and polarization imaging analysis.

Resolution of electrogenic steps coupled to conversion of cytochrome c oxidase from the peroxy to the ferryl-oxo state.

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl-oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl-oxo-to-oxidized (F --> O) transition of the enzyme [Zaslavsky, D., et al. (1993) FEBS Lett. 336, 389-393]. As in the case of the F --> O transition, the electric response associated with photoreduction of P to F includes a rapid KCN-insensitive electrogenic phase with a tau of 40-50 microseconds (reduction of heme a by CuA) and a multiphasic slower part; this part is cyanide-sensitive and is assigned to vectorial transfer of protons coupled to reduction of oxygen intermediate in the binuclear center. The net KCN-sensitive phase of the response is approximately 4-fold more electrogenic than the rapid phase, which is similar to the characteristics of the F --> O electrogenic transition and is consistent with net transmembrane translocation of two protons per electron, including vectorial movement of both "chemical" and "pumped" protons. The protonic part of the P --> F electric response is faster than in the F --> O transition and can be deconvoluted into three exponential phases with tau values varying for different samples in the range of 0.25-0.33, 1-1.5, and 6-7.5 ms at pH 8. Of these three phases, the 1-1.5 ms component is the major one contributing 50-60%. The P --> F conversion induced by single electron photoreduction of the peroxy state as studied in this work is several times slower than the P --> F transition resolved during oxidation of the fully reduced oxidase by molecular oxygen. The role of the CuB redox state in controlling the rate of P --> F conversion of heme a3 is discussed.

Martin Rodbell (1925-1998).

Martin Rodbell (1925-1998).

Sugar Transporters in Polarized Epithelial Cells.

Sugar transporters are integral membrane proteins responsible for the transport of sugars across the cellular membranes. They play important roles in the transfer of sugars across different compartments separated by epithelia in the body. Preferential localization of them in specific plasma membrane domains of epithelial cells is crucial in the vectorial transfer of sugars. Modification of the transporter genes with the molecular biology techniques and visualization of the expressed gene products by immunohistochemical methods have shed light on the molecular mechanism underlying the specific localization of the transporter molecules.

The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation

Ferritins uniquely direct the vectorial transfer of hydrated Fe(II)/Fe(III) ions to a condensed ferric phase in the central cavity of the soluble protein. Secondary, tertiary and quaternary structure are conserved in ferritin, but only five amino acid residues are conserved among all known ferritins. The sensitivity of ferroxidation rates to small differences in primary sequence between ferritin subunits that are cell-specifically expressed or to the conservative replacement of the conserved tyrosine 30 residue was demonstrated by examining recombinant (frog) H-type (red blood cell predominant) and M-type subunit (liver predominant) proteins which are both fast ferritins; the proteins form two differently colored Fe(III)-protein complexes absorbing at 550 nm or 650 nm, respectively. The complexes are convenient reporters of Fe(III)-protein interaction because they are transient in contrast to the Fe(III)-oxy complexes measured in the past at 310–420 nm, which are stable because of contributions from the mineral itself. The A650-nm species formed 18-fold faster in the M-subunit protein than did the 550-nm species in H-subunit ferritin, even though all the ferroxidase residues are the same; the Vmax was fivefold faster but the Hill coefficents were identical (1.6), suggesting similar mechanisms. In H-subunit ferritin, substitution of phenylalanine for conserved tyrosine 30 (located in the core of the subunit four-helix bundle) slowed ferroxidation tenfold, whereas changing surface tyrosine 25 or tyrosine 28 had no effect. The Fe(III)-tyrosinate was fortunately not changed by the mutation, based on the resonance Raman spectrum, and remained a suitable reporter for Fe(III)-protein interactions. Thus, the A550/650 nm can also report on post-oxidation events such as transport through the protein. The impact of Y30F on rates of formation of Fe(III)-protein complexes in ferritin, combined with Mossbauer spectroscopic studies that showed the parallel formation of multiple Fe(III) postoxidation species (three dinuclear oxy and one trinuclear oxy species) (A. S. Periera et al., Biochemistry 36 : 7917–7927, 1997) and the loss of several of the multimeric Fe(III) post-oxidation species in a Y30F alteration of human recombinant H-ferritin (E. R. Bauminger et al., Biochem J. 296 : 709–719, 1993), indicate that at least one of the pathways for Fe oxidation/transfer in ferritin is through the center of the four-helix bundle and is influenced by structural features dependent on tyrosine 30.

Electrogenic reduction of the primary electron donor P700 + in photosystem I by redox dyes

The kinetics of reduction of the photo-oxidized primary electron donor P700 + by redox dyes N,N,N′, N′-tetramethyl- p-phenylendiamine, 2,6-dichlorophenol-indophenol and phenazine methosulfate was studied in proteoliposomes containing Photosystem I complexes from cyanobacteria Synechocystis sp. PCC 6803 using direct electrometrical technique. In the presence of high concentrations of redox dyes, the fast generation of a membrane potential related to electron transfer between P700 and the terminal iron-sulfur clusters F A/F B was followed by a new electrogenic phase in the millisecond time domain, which contributes approximately 20% to the overall photoelectric response. This phase is ascribed to the vectorial transfer of an electron from the redox dye to the protein-embedded chlorophyll of P700 +. Since the contribution of this electrogenic phase in the presence of artificial redox dyes is approximately equal to that of the phase observed earlier in the presence of cytochrome c 6, it is likely that electrogenic reduction of P700 + in vivo occurs due to vectorial electron transfer within RC molecule rather than within the cytochrome c 6-P700 complex.

Vectorial transfer of electronic energy in rod-like ruthenium–osmium dinuclear complexes

Dinuclear complexes containing ruthenium and osmium-based bis-terpyridyl chromophoric termini are prepared and their luminescence properties investigated. The two chromophoric units are connected by 1,4-phenylenes only, or phenylene and bicyclooctane spacers. In this way complete control of the geometry of the dinuclear complexes is achieved and these rigid species resemble molecular rods or girders featuring intermetal distances in the range 11–24 A. The Ru→Os transfer of electronic excitation is energetically allowed and we have studied the effect on this process both of the intermetal separation and the electronic properties of the spacers. The main conclusions are that the phenylene spacers are very efficient in transmitting the intermetal electronic communication but an important role is also played by the spatial localization of the metal-to-ligand charge-transfer excited states involved in the excitation-transfer process.

Merck Frosst award lecture 1995. La conference Merck Frosst 1995. Structural studies of lipoproteins and their apolipoprotein components.

Lipid transport processes via the circulatory system of animals are a vital function that utilizes highly specialized lipoprotein complexes. These complexes of protein and lipid impart solubility to otherwise insoluble lipids. The apoprotein components of lipoprotein complexes serve to stabilize the lipid components and modulate particle metabolism and function as ligands for receptor-mediated endocytosis of lipoproteins. We have used an insect (Manduca sexta) model system for studies of lipid transport. In this system, flight activity elicits a dramatic increase in the demand for glycerolipid fuel molecules by flight muscle tissue. These lipids are mobilized from a storage organ and transported through the hemolymph (blood) to the flight muscle by the lipoprotein, lipophorin. This system possesses the unique property that lipids are loaded onto pre-existing high density lipophorin through the action of a lipid transfer particle (LTP). LTP is a high molecular weight hemolymph component that facilitates net vectorial lipid transfer from fat body tissue to lipophorin. The increase in lipid content of the lipoprotein induces association of a low molecular weight amphipathic exchangeable apolipoprotein, apolipophorin III (apoLp-III). ApoLp-III is a 18 kDa protein that normally exists as a water-soluble monomeric hemolymph protein. The structural properties of apoLp-III have been investigated by X-ray crystallography. ApoLp-III from Locusta migratoria adopts a five helix bundle conformation wherein each of the amphipathic helices orients with its hydrophobic face directed toward the interior of the bundle. It has been hypothesized that lipid association requires a dramatic conformational change wherein the helix bundle opens about putative hinge domains located in the loops between helices. The data accumulated support the concept that apoLp-III is a member of the broad class of exchangeable apolipoproteins and structural information learned from this system is directly applicable to analogous proteins in higher organisms.

Developmental and comparative ultrastructure of glandular hairs in the two Urticaceae. I. Urtica dioica

Secretion produced by glandular hairs is deposited mainly in the periplasmic space of the head cells. It stains intensely for both proteins and polysaccharides. The ultrastructure of meristematic, differentiating, mature and senescent head cells as well as the stalk and basal cells has been described in comparison to that in other cell types of the leaf. The specific features of the head cells are the proliferation of the granular endoplasmic reticulum as well as the multiplication of the dictyosomes and mitochondria during transition to the secretion stage. However, the frequency of dictyosomes varies among secreting hairs. The ER produces neither secretory nor transition vesicles and does not anastomose with the plasmalemma. In the absence of transition vesicles, the transport of secretory proteins and enzymes of polysaccharide synthesis from the ER to dictyosomes apparently includes the cytosolic step. Dictyosomes, though not appearing hypersecretory, produce two types of smooth secretory vesicles generated by the trans Golgi reticulum. The vectorial transfer of prosecretion and membranes across the dictyosome stack proceeds via the transport (shuttle) vesicles. It is, therefore, concluded that exocytosis of smooth secretory Golgi vesicles is the sole mechanism of release of both proteins and polysaccharides. Coated vesicles occasionally seen near the plasmalemma are likely to be involved in the endocytotic membrane retrieval. The secretion product disappears during senescence of the hairs and the secretory cells undergo vacuolation by means of local autophagy.

Influence of hydroxylation and conjugation in cross-inhibition of bile acid transport across the human trophoblast basal membrane

Taurocholate (TC) transport across the basal plasma membrane of the human trophoblast is a carrier-mediated process, whose specificity is probably not restricted to TC. The aim of this work was to gain further insight into the role of hydroxylation and conjugation in the behavior of the carrier system vs. bile acid (BA) species. Radiolabeled TC transport by basal plasma membrane (BPM) vesicles obtained from human term placenta was measured by a rapid filtration technique. Glycocholate (GC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC) inhibited TC binding to BPM. These bile acids complete with TC for the binding sites. Symmetry properties for GC- and TCDC-induced inhibition of TC transport was found in experiments where GC or TCDC were at the cis-side of the membrane (uptake and efflux experiments). GC and TCDC-induced inhibition seems to be of mixed type. By contrast, TDC was observed to affect TC transport differently, depending on whether the experiments addressed uptake or efflux. At the intracellular side of the membrane (uptake), TDC induced a marked increase in both V max and K t . However, at the fetal side (efflux) a significant reduction in both V max and K t was found. In spite of these peculiarities, the values for K i were very close for GC, TCDC and TDC at the intracellular side but not at the fetal side, where the decreasing order for K i was GC > TCDC > TDC . TC uptake by BPM vesicles was not modified in the presence of a wide range of estrone sulfate concentrations (0.002–1.0 mM). In summary, these results indicate that a particular bile acid molecular structure is necessary for steroid-related compounds to interact with the bile acid carrier located in BPM. They also suggest that changes in the number and position of hydroxy groups, as well as in the amino-acid moiety in amidated bile acids modify the behavior of the carrier, which may play an important role in the net vectorial transfer of bile acids across the placenta.

Patterns of mitochondrial sorting in yeast zygotes.

Inheritance of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae is usually biparental. Pedigree studies of zygotic first buds indicate limited mixing of wild-type (p+) parental mtDNAs: end buds are frequently homoplasmic for one parental mtDNA, while heteroplasmic and recombinant progeny usually arise from medial buds. In crosses involving certain petites, however, mitochondrial inheritance can be uniparental. In this study we show that mitochondrial sorting can be influenced by the parental mtDNAs and have identified intermediates in the process. In crosses where mtDNA mixing is limited and one parent is prelabeled with the matrix enzyme citrate synthase 1 (CS1), the protein freely equilibrates throughout the zygote before the first bud has matured. Furthermore, if one parent is p0 (lacking mtDNA), mtDNA from the p+ parent can also equilibrate; intracellular movement of mtDNA is unhindered in this case. Surprisingly, in zygotes from a p0 CS1+ x p+ CS1- cross, CS1 is quantitatively translocated to the p+ end of the zygote before mtDNA movement; subsequently, both components equilibrate throughout the cell. This initial vectorial transfer does not require respiratory function in the p+ parent, although it does not occur if that parent is p-. Mouse dihydrofolate reductase (DHFR) present in the mitochondrial matrix can also be vectorially translocated, indicating that the process is general. Our data suggest that in zygotes mtDNA movement may be separately controlled from the movement of bulk matrix constituents.

A turbidimetric assay of lipid transfer activity

A novel method to assay insect plasma lipid transferparticle (LTP) activity has been developed that employs insect high density lipophorin (HDLp) and human low density lipoprotein (LDL) as donor/acceptor substrate particles. At a 3:1 or greater HDLp:LDL protein ratio, LTP-mediated net vectorial transfer of diacylglycerol from lipophorin to LDL produces destabilized LDL particles that aggregate, causing sample turbidity. Turbidity was measured spectrophotometrically as a function of absorbance at 340 nm. After an initial lag phase, lipoprotein sample turbidity increased as a function of reaction time and LTP concentration. Saturation was observed at longer times or higher LTP concentrations, indicating that a reaction end point had been reached. As the substrate HDLp concentration was increased relative to LDL, a saturable increase in LTP-induced lipoprotein sample turbidity was observed. When the LDL concentration was increased relative to HDLp, however, there was an initial production of turbidity but at higher concentrations the sample did not develop turbidity. Reaction progress was also dependent on temperature over the range 0–37°C. Taken together the results are consistent with the concept that LTP-mediated diacylglycerol transfer from HDLp to LDL creates unstable product LDL particles that aggregate. The assay method is advantageous because it employs relatively abundant, natural lipoprotein substrates, does not require prelabeling of donor lipid particles with radioactive or fluorescent lipids, and does not require separation of donor and acceptor after incubation. This is the first description of a lipid transfer assay that can be measured spectrophotometrically.

Insect lipid transfer particle can facilitate net vectorial lipid transfer via a carrier-mediated mechanism.

The mechanism of facilitated lipid transfer by insect or mammalian plasma lipid transfer proteins has not been elucidated. Transfer catalysts may act as carriers of lipid between donor and acceptor lipoproteins or, alternatively, transfer may require formation of a ternary complex. This study was designed to determine if Manduca sexta hemolymph lipid transfer particle (LTP) can facilitate net vectorial transfer of lipid without concomitant contact between donor and acceptor lipoproteins and LTP. M. sexta [3H]diacylglycerol-high density lipophorin-larval ([3H]DAG-HDLp-L) and human low density lipoprotein (LDL) were covalently bound to Sepharose matrices and packed into separate columns. In incubations lacking LTP, greater than 98% of the recovered DAG remained associated with HDLp-L. An unrelated hemolymph storage protein, arylphorin, was unable to catalyze the transfer of DAG between solid-phase lipoproteins. Facilitated transfer of DAG from HDLp-L to LDL was observed when LTP was circulated between the columns. Under these conditions, facilitated transfer occurred at a rate of 2.24 ng of DAG/h (versus 0.16 microgram of DAG/h in the control), and after 16 h greater than 26% of recovered labeled DAG was transferred to LDL. This corresponds to a 14-fold rate enhancement induced by LTP. The LTP-specific transfer of DAG between physically separated lipoproteins demonstrates the ability of LTP to facilitate net lipid transfer via a carrier-mediated mechanism in the absence of a ternary complex involving donor, acceptor, and catalyst. In experiments aimed at assessing the relative contribution of ternary complex formation to DAG transfer, acceptor LDL was circulated with HDLp-L remaining immobilized. Under these conditions, LTP induced a 13-fold rate enhancement from 1.3 to 16.3 micrograms of DAG/h. The similar rate enhancements observed with both lipoproteins bound and only donor bound suggest the overall contribution of ternary complex formation to facilitated lipid transfer is insignificant. The described system should prove useful in mechanistic studies of other transfer proteins as well as studies of transfer of other lipids.