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PubMed1975

The glyceraldehyde 3-phosphate and glycerate 3-phosphate shuttle and carbon dioxide assimilation in intact spinach chloroplasts.

The regulation of CO(2) assimilation by intact spinach (Spinacia oleracea) chloroplasts by exogenous NADP-linked nonreversible d-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) was investigated. This dehydrogenase mediated a glyceraldehyde 3-phosphate/glycerate 3-phosphate shuttle for the indirect transfer of NADPH from chloroplast to the external medium. The rate of NADPH formation in the medium reflected glyceraldehyde 3-phosphate efflux from the chloroplast. Increasing enzyme concentrations stimulated NADP reduction and, in turn, CO(2) fixation. Pyrophosphate increased CO(2) fixation by apparently inhibiting glyceraldehyde 3-phosphate efflux. Increasing the glycerate 3-phosphate concentration above 0.1 mm stimulated glyceraldehyde 3-phosphate efflux but inhibited CO(2) fixation. Addition of up to 0.5 mm orthophosphate enhanced both glyceraldehyde 3-phosphate efflux and CO(2) fixation while each was inhibited by higher orthophosphate concentrations. The mechanism by which the extent of glyceraldehyde 3-phosphate efflux regulated the rate of CO(2) fixation in chloroplasts was discussed.

Journal Article↗
PubMed1989

Calvin-Benson Cycle Enzymes in Guard-Cell Protoplasts from Vicia faba L: Implications for the Greater Utilization of Phosphoglycerate/Dihydroxyacetone Phosphate Shuttle between Chloroplasts and the Cytosol.

Activities of Calvin-Benson cycle enzymes were found in protoplasts of guard cells from Vicia faba L. The activities of NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD) and ribulose-1,5-bisphosphate carboxylase (RuBPC) were 2670 and 52 micromoles per milligrams chlorophyll per hour, respectively. Activities of NADP-GAPD and RuBPC in guard cells were increased by red light illumination, and the light activations were inhibited completely by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Enzymes related to the Calvin-Benson cycle such as 3-phosphoglycerate kinase (PGAK), triose phosphate (TP) isomerase, and fructose-1,6-bisphosphatase (FBPase) were shown to be present in guard-cell chloroplasts. From these results, we conclude that the photosynthetic carbon reduction pathway is present in guard-cell chloroplasts of Vicia faba. We compared these enzyme activities in guard cells with those in mesophyll cells. The activities of NADP-GAPD and PGAK were more than several-fold higher and that of TP isomerase was much higher in guard-cell chloroplasts than in mesophyll chloroplasts. In contrast, activities of RuBPC and FBPase were estimated to be roughly half of those in mesophyll chloroplasts. High activities of PGAK, NAD-GAPD, and TP isomerase were found in fractions enriched in cytosol of guard cells. Illumination of guard-cell protoplasts with red light increased the cellular ATP/ADP ratio from 5 to 14. These results support the interpretation that guard cells utilize a shuttle system (e.g. phosphoglycerate [PGA]/dihydroxyacetone phosphate [DHAP] shuttle) for an indirect transfer of ATP and reducing equivalents from chloroplasts to the cytosol.

Journal Article↗
PubMed2001

Performance of asbestos fibre counting laboratories by transmission electron microscopy.

In France, the owners of buildings have been obliged since February 1996 to ascertain whether asbestos has been incorporated into surfacing materials, insulation products or false ceilings. In certain circumstances, there is also a requirement to measure the airborne asbestos fibre concentration. Three years (1996-1998) of asbestos fibre count reporting are evaluated for the proficiency testing scheme organized in France to evaluate the performance of laboratories using an indirect-transfer transmission electron microscopy procedure to measure the airborne asbestos fibre concentration. Each year eight filters are distributed to each participating laboratory. These filters are obtained by filtering a suspension containing chrysotile or amphibole fibres. In 1996, 36% of the laboratories were rated 1 (the best performers; i.e. those providing counts close to the reference value). Performance improved appreciably in the last round where 85% of the laboratories were rated 1.

Air Pollution, Indoor↗
PubMed1988

Molecular characterization of serologic recognition sites in the human HLA-A2 molecule.

The localization of the amino acid residues involved in the serologic specificity of the HLA-A2 molecule has been investigated using a combination of site-directed mutagenesis, DNA-mediated gene transfer, indirect immunofluorescence and flow cytometry techniques. Synthetic oligonucleotides were designed to introduce individual and combined amino acid substitutions in both the alpha 1 (positions 9, 43, and the highly polymorphic cluster of residues from aa 62 to 83) and alpha 2 (positions 107, 152, and 156) domains to investigate the effect of the specific mutation on the recognition of the molecule at the surface of transfected human and mouse cell lines by a panel of mAb that recognize monomorphic or polymorphic determinants in MHC class I molecules. At least three non-overlapping serologic epitopes were identified. Mutations in the highly polymorphic region at aa 62 to 66 completely eliminated binding of mAb MA2.1 (A2/B17 cross-reactive). Mutation at position 107 resulted in complete loss of binding of the A2/Aw69-specific mAb PA2.1 and MA2.2 and partial loss of mAb BB7.2 binding. The recognition by other allotypic mAbs was not affected by these mutations and they therefore represent at least a third serologic epitope. Mutations at positions 152 and 156, known to be important for T cell recognition, did not affect serologic recognition. Introduction of residues of HLA-B7 origin in the polymorphic segment spanning aa 70 to 80 created a molecule carrying the -Bw6 supertypic determinant as demonstrated by mAb SFR8-B6 binding.

Amino Acid Sequence↗
PubMed1998

Direct/indirect band placement.

This article describes orthodontic band placement in alginate impressions. The fitting of orthodontic bands at the chair and the indirect transfer of the bands to the impression influences the fit and function of the preventive or interceptive orthodontic appliance being fabricated. Some of the problems associated with the transfer of bands to impressions will be presented. In addition, a discussion of methods to eliminate these problems is included.

Alginates↗
PubMed1996

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.

Animals↗
PubMed2001

Protein standardization IV: Value transfer procedure for the assignment of serum protein values from a reference preparation to a target material.

A new approach for the assignment of values to serum proteins in a target material using a reference preparation has been developed. The procedure describes the general as well as the practical principles involved in the value assignment (with examples). Two models have been developed: 1) The direct value transfer between serum matrices and 2) the indirect value transfer from a pure protein preparation to a serum protein material. The necessary mathematical equations are developed and explained. The data reduction and statistical evaluation are discussed. The practical procedure (the transfer protocol) is based on six dilutions of the reference preparation assayed together with six dilutions of the target material. In this way imprecision is reduced and the proportionality of the two materials (i.e. the presence or absence of matrix effects) can be assessed directly by evaluating a single regression plot. If no matrix effects are found, the regression line will pass through zero with a slope equal to the ratio of the concentrations of the two materials. The transfer protocol is based on a multiple point value assignment obtained by several measurements a day repeated on several days, an important prerequisite being that all reconstitutions and dilutions are controlled by weighing.

Blood Proteins↗
PubMed2004

Study of oral tolerance and its indirect effects in adoptive cell transfer experiments.

Parenteral exposure to antigens to which oral tolerance had been previously induced results in the inhibition of immune responses to other unrelated antigens. Herein we tested whether indirect effects of oral tolerance could be adoptively transferred. Anti-Ova- and antihemoglobin-specific responsiveness as well as oral tolernace to Ova were transferred to irradiated, but not to normal, nonirradiated recepients. Irradiation, thus, facilitated adoptive transfer of oral tolerance. However, the inhibitory (indirect) effects upon the unrelated immunogen were not adoptively transferred, even to irradiated recepients. In addition, we studied adoptively transferred CFSE-labeled spleen cells by flow cytometry in recipient spleen, inguinal lymph nodes, and bone marrow, both in irradiated and nonirradiated recipients, 1, 3, or 5 days after cell transfer. Comparing the percent and absolute number of CFSE-labeled cells in each organ displayed significant differences in the dynamics of decay of adoptively transferred cells from tolerant or immune donors.

Administration, Oral↗
PubMed1993

[Luminescence and detection in liquid chromatography. II--Energy transfer from a stimulated molecule: indirect potentialization or sensitized luminescence].

Under the words of sensitized luminescence, the processes who include the transfers of energy from a donor to an acceptor are described. They are to the origin of new possibilities of detection in liquid chromatography by emission or inhibition of phosphorescence and fluorescence. These news technologies are illustrated by examples in pharmaceutic, biologic or food applications.

Chromatography, Liquid↗
PubMed1988

Intracellular localization of beta-glucuronidase in fibroblasts after direct transfer from macrophages.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.

Cell Communication↗
PubMedDate not supplied

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles.

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.

Dose-Response Relationship, Drug↗
PubMed1993

Staphylococcus epidermidis--hospital epidemiology and the detection of methicillin resistance.

Infections in immunocompromised patients and in patients with indwelling prosthetic devices are often caused by hospital strains of Staphylococcus epidermidis resistant to methicillin. Tests for the detection of methicillin resistance, indicating resistance to all beta-lactam antibiotics, were evaluated in order to define a suitable screening test. A broth tube breakpoint test with a large inoculum, 10(7) colony forming units (cfu), gave the highest recovery of resistant strains. False resistance due to hyperproduction of beta-lactamase was excluded. The results correlated completely with the detection of the resistance gene, mecA, by the polymerase chain reaction. In 2/3 of the resistant strains tested the expression of the methicillin resistance was heterogeneous, only one cell in 10(2) to 10(4) expressed the resistance within 72 h in both. In broth screening tests an inoculum of at least 10(6) cfu therefore was required to detect all resistant strains within 24 h. Using agar dilution, 48 h incubation must be considered. In disc diffusion tests reliable results were obtained after only 16 h of incubation when discs containing cephradine 5 and 30 micrograms, oxacillin 1 microgram or cephalexin 30 micrograms were used, and the first disc is recommended for routine work. The epidemiology of S. epidermidis strains resistant to ciprofloxacin and/or gentamicin was studied in an isolation unit for patients undergoing bone marrow transplantation. Antibiograms and plasmids were used for typing and 31 such strains were found. Of 54 staff members 10 were colonized in the nares only, two in the nares and perineum and one in the nares and stool. In ambient air and on the clothes of staff a few of the strains predominated quantitatively. These strains colonized the skin of some of the patients who seemed to be the main dispersers. Possible routes of cross-infection were indirect contact transfer via the hands and clothes of staff (82% of the clothes were contaminated), and direct as well as indirect airborne transmission. To study the effects of chlorhexidine on skin bacteria, ten nurses washed one arm with chlorhexidine-detergent every morning for 3 weeks; the other arm served as control. The depression of the normal skin flora did not lead to a colonization with more antibiotic-resistant hospital strains. During the wash period the counts of antibiotic-resistant S. epidermidis on the treated arms were significantly reduced compared with the control arms, as also were the number of different strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1975

Airborne infection in a fully air-conditioned hospital. IV. Airborne dispersal of Staphylococcus aureus and its nasal acquisition by patients.

Studies in a newly built hospital furnished with complete air conditioning where most of the patients are nursed in 6-bed rooms showed that the transfer of air from one patient room to another was very small, especially when there was substantial flow of air in a consistent direction between the patient rooms and the corridor, and that the direct transfer of airborne particles was even less. There was, however, no evidence of any reduction in the rates of nasal acquisition of Staphylococcus aureus compared with those to be found in naturally ventilated hospitals. The numbers of Staph. aureus found in the air of a given room that appeared to have originated from patient carriers in other rooms were many times greater than could be accounted for by direct airborne transfer. Although there was evidence that many carriers were not detected, detailed study showed that this excess transfer to the air of other rooms was genuine. It seems probable on the basis of investigations in this hospital and elsewhere that this excess transfer occurs indirectly, through dispersal from the clothing of the nursing and medical staff into the air of another room of strains with which their outer clothes have become contaminated while dealing with patients. Reduction in direct airborne transfer of micro-organisms from one room to another, whether by ventilation or other means, can only be of clinical advantage if transfer by other routes is, or can be made, less than that by the direct airborne route.

Adult↗
PubMed1990

The diagnosis of multiple personality disorder based on subtle dissociative signs.

Patients with multiple personality disorder (MPD) often present with subtle dissociative signs and symptoms rather than with the clear-cut, overt dissociative signs and symptoms usually associated with MPD. Because most MPD patients usually conceal their condition, there are only limited periods in their lives when they show florid, overt symptoms that can be easily diagnosed. During these other times, MPD patients often show subtle dissociative signs in their affects, thoughts, memories, behaviors, object relations, and transferences. These indirect manifestations of MPD are evidence of the patient's dissociative pathology and occur when their alters influence each other, partly emerge, or subtly shift. A familiarity with these subtle dissociative signs would allow the therapist to be aware of what MPD looks like when it is camouflaged and expressed indirectly. A recognition of these indirect manifestations would enable the clinician to suspect the diagnosis, explore the possible alters, and diagnose the patient's underlying dissociative pathology when present.

Adult↗
PubMed2003

Mechanistic insights revealed through characterization of a novel chromophore in selenophosphate synthetase from Escherichia coli.

The incorporation of selenium into specific proteins and tRNAs requires selenophosphate (SePO3), whose formation is catalyzed by selenophosphate synthetase. In a Mg/ATP-dependent reaction, selenophosphate synthetase catalyzes the phosphorylation of selenide to yield AMP, inorganic phosphate, and SePO3. In this report, a previously unrecognized chromophore covalently attached to selenophosphate synthetase is characterized. The UV/Vis spectrum of selenophosphate synthetase has a feature centered at 315 nm that is irreversibly destroyed by alkylation. Moreover, addition of Zn2+, which is known to inhibit selenophosphate synthetase, reversibly quenches the 315 nm absorption. Since Zn2+ is known to bind to Cys17, these data strongly suggest that this residue participates in the 315 nm absorption. Upon incubation with both Mg2+ and ATP, the lambda(max) of the chromophore shifts to 340 nm, and it is shown that the shift requires binding of nucleotide having a hydrolyzable gamma-phosphoryl group. These data indicate that either the chromophore is directly involved in phosphoryl transfer or indirectly reflects a phosphorylation-dependent conformational change in selenophosphate synthetase. This work provides the first spectroscopic handle on catalytic steps associated with SePO3 synthesis, which will be used to study the molecular structure of the chromophore and its role in the catalytic mechanism of selenophosphate synthetase.

Adenosine Triphosphate↗
PubMed2000

Adoptively transferred gamma delta T cells indirectly regulate murine graft-versus-host reactivity following donor leukocyte infusion therapy in mice.

The purpose of this study was to determine whether gamma delta T cells were able to regulate graft-vs-host (GVH) reactivity mediated by alpha beta T cells in murine recipients transplanted with MHC-mismatched marrow grafts. Studies were conducted using ex vivo-activated gamma delta T cells because this was a more clinically relevant strategy, and these cells have been shown to be capable of facilitating alloengraftment without causing GVH disease (GVHD). Coadministration of activated gamma delta T cells and naive alpha beta T cells at the time of bone marrow transplantation (BMT) significantly exacerbated GVHD when compared with naive alpha beta T cells alone. In contrast, when the administration of naive alpha beta T cells was delayed for 2 wk post-BMT, survival was significantly enhanced in mice transplanted with BM plus activated gamma delta T cells vs those given marrow cells alone. Mitigation of GVHD by activated gamma delta T cells occurred only at high doses (150 x 106) and was a unique property of gamma delta T cells, as activated alpha beta T cells were incapable of ameliorating the subsequent development of GVHD. Protection from GVHD was not due to the direct inhibition of naive alpha beta T cells by gamma delta T cells. Rather, gamma delta T cells mediated this effect indirectly through donor BM-derived alpha beta T cells that acted as the proximate regulatory population responsible for the decrease in GVH reactivity. Collectively, these data demonstrate that activated gamma delta T cells are capable of modulating the ability of MHC-incompatible nontolerant alpha beta T cells to cause GVHD after allogeneic BMT.

Adoptive Transfer↗