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Biomedical subjects

Z Gombos

Publications and source records attributed to Z Gombos.

At least 37 records · Page 2Linked to original sources
PubMed1996

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.

Calorimetry, Differential Scanning↗
PubMed1996

Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.

There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes.

Journal Article↗
PubMed1996

Characterization of the Fad12 mutant of Synechocystis that is defective in delta 12 acyl-lipid desaturase activity.

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for delta 12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the delta 12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the delta 12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.

Amino Acid Sequence↗
PubMed1996

Antisense RNA inhibition of the putative vacuolar H(+)-ATPase proteolipid of Dictyostelium reduces intracellular Ca2+ transport and cell viability.

Transport of Ca2+ via a P-type pump into the contractile vacuole of Dictyostelium discoideum appears to be facilitated by vacuolar proton (V-H+) ATPase activity. To investigate the involvement of the V-H(+)-ATPase in this process using molecular techniques, we cloned a cDNA (vatP) encoding the putative proteolipid subunit of this enzyme. The deduced protein product of this cDNA is composed of 196 amino acids with a calculated M(r) of 20,148 and the primary structure exhibits high amino acid sequence identity with V-H(+)-ATPase proteolipids from other organisms. vatP is a single-copy gene and it produces one approximately 900 nt transcript at relatively constant levels during growth and development. Attempts to disrupt the endogenous gene using vatP cDNA were unsuccessful. But, expression of vatP antisense RNA reduced the levels of vatP message and V-H(+)-ATPase activity by 50% or more. These antisense strains grew and developed slowly, especially under acidic conditions, and the cells seemed to have difficulty forming acidic vesicles. During prolonged cultivation, all of the antisense strains either reverted to a wild-type phenotype or died. Thus in Dictyostelium, unlike yeast, the V-H(+)-ATPase seems to be indispensable for cell viability. When different antisense strains were analyzed for Ca2+ uptake by the contractile vacuole, they all accumulated less Ca2+ than control transformants. These results are consistent with earlier pharmacological studies which suggested that the V-H(+)-ATPase functions in intracellular Ca2+ transport in this organism.

Amino Acid Sequence↗
PubMed1995

Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants.

Using tobacco plants that had been transformed with the cDNA for glycerol-3-phosphate acyltransferase, we have demonstrated that chilling tolerance is affected by the levels of unsaturated membrane lipids. In the present study, we examined the effects of the transformation of tobacco plants with cDNA for glycerol-3-phosphate acyltransferase from squash on the unsaturation of fatty acids in thylakoid membrane lipids and the response of photosynthesis to various temperatures. Of the four major lipid classes isolated from the thylakoid membranes, phosphatidylglycerol showed the most conspicuous decrease in the level of unsaturation in the transformed plants. The isolated thylakoid membranes from wild-type and transgenic plants did not significantly differ from each other in terms of the sensitivity of photosystem II to high and low temperatures and also to photoinhibition. However, leaves of the transformed plants were more sensitive to photoinhibition than those of wild-type plants. Moreover, the recovery of photosynthesis from photoinhibition in leaves of wild-type plants was faster than that in leaves of the transgenic tobacco plants. These results suggest that unsaturation of fatty acids of phosphatidylglycerol in thylakoid membranes stabilizes the photosynthetic machinery against low-temperature photoinhibition by accelerating the recovery of the photosystem II protein complex.

Acclimatization↗
PubMed1994

Structure and interactions of phycocyanobilin chromophores in phycocyanin and allophycocyanin from an analysis of their resonance raman spectra.

Raman spectra of phycocyanobilin, phycocyanin, and allophycocyanin were obtained at resonance with their visible and near-UV transitions. These spectra were empirically assigned with the help of 14N- and 15N-isotopic substitutions and comparisons with resonance Raman spectra of phycoerythrin. These results confirm the previously suggested assignment of a conformation-sensitive band around 1239-1246 cm-1 to a mode involving nu CmH and nu CN coordinates. Computer-assisted decomposition of the complex, conformation-sensitive 1580-1670-cm-1 region yielded five components that we labeled I-V. The previously described spectral changes observed upon monomerization and denaturation in resonance Raman spectra of phycocyanin and allophycocyanin essentially arise from changes in the relative intensities of these components. Component I (around 1649-1651 cm-1) and component III (1621-1624 cm-1) originate predominantly from nu C=C at C15 of the chromophore. Their relative intensity ratio reflects the relative amounts of C15-Z-anti and C15-Z-syn methine bridge conformations, respectively. Component II (1633-1638 cm-1) is ascribed to a nu C=C mode of pyrrole rings; it is not sensitive to the chromophore conformation. Component IV is also conformation-insensitive and originates from nu C=N and nu C=C coordinates, most likely from ring C. Component V (1591-1594 cm-1) involves a nu C=N coordinate in ring D, coupled to a nu C=C coordinate of the C15 methine bridge. The implications of the present assignments on those of resonance Raman active modes of phytochrome are discussed. A consistent set of correlations between chromophore conformations and resonance Raman data is obtained for both phycobiliproteins and phytochrome.

Cyanobacteria↗
PubMed1994

The recovery of photosynthesis from low-temperature photoinhibition is accelerated by the unsaturation of membrane lipids: a mechanism of chilling tolerance.

In a previous study of mutants in fatty-acid desaturation of Synechocystis PCC6803, it was demonstrated that the photoinhibition of photosynthesis at low temperature in vivo is tolerated by cells as a result of the unsaturation of glycerolipids of thylakoid membranes. Since the extent of photoinhibition of photosynthesis in vivo depends on a balance between the photoinduced inactivation and the recovery from the photoinhibited state, an examination was made of the effects of the unsaturation of membrane lipids on these processes. It appears that the unsaturation of the membrane lipids does not affect the inactivation process but accelerates the recovery process and, moreover, that the apparent increase in the photoinhibition in vivo of photosynthesis at low temperature is caused by a depressed rate of recovery at low temperature.

Chloramphenicol↗
PubMed1994

Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress.

The contribution of the unsaturation of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress was studied in a transgenic cyanobacterium. Anacystis nidulans R2-SPc was transformed with the desA gene, which encodes the Delta12-desaturase that desaturates the fatty acids of membrane lipids in Synechocystis PCC6803. The transformant acquired the ability to introduce a second double bond into palmitoleic and oleic acids. The transformation enhanced the tolerance of the photosynthetic machinery to chilling stress but it had no detectable effect on the ability to tolerate heat stress. The transformation itself did not have any effect on photosynthetic activity. These results imply that an increase in the unsaturation of membrane lipids enhances the tolerance of the photosynthetic machinery toward chilling stress but not toward heat stress and that such an increase does not affect photosynthesis within the range of physiological temperatures.

Journal Article↗
PubMed1992

Unsaturation of fatty acids in membrane lipids enhances tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature photoinhibition.

Effect of the unsaturation of fatty acids in the glycerolipids of thylakoid membranes on low-temperature photoinhibition of photosynthesis was studied by mutation and transformation of the cyanobacterium Synechocystis PCC6803. When grown at 34 degrees C, the wild type contained mono-, di-, and triunsaturated lipids; a mutant, designated Fad6, contained mono- and diunsaturated lipids; and a transformant of Fad6, with a disrupted gene for desaturation and designated Fad6/desA::Kmr, contained only monounsaturated lipids. Fad6/desA::Kmr was the most susceptible among these strains to low-temperature photoinhibition of photosynthesis, whereas Fad6 and the wild type were apparently indistinguishable in terms of sensitivity to photoinhibition. This result suggests that the presence of diunsaturated fatty acids is important in protecting against low-temperature photoinhibition. The photoinhibition at room temperature, although much less significant than that at low temperature, was also affected by the unsaturation of fatty acids. By contrast, the photosynthetic transport of electrons, measured at various temperatures, was not affected by changes in extent of fatty acid unsaturation.

Cold Temperature↗
PubMed1992

Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin.

Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa. Heat stress at 42.5 degrees C appeared to be the optimum temperature for HSP formation in cells grown at 30 degrees C. The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h. The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli. To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity. There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942. Moreover, the purified HSP64 cross-reacted to anti-E. coli GroEL antibody. To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.

Amino Acid Sequence↗
PubMed1990

Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation.

The sensitivity (or tolerance) of plants to chilling determines their choice of natural habitat and also limits the worldwide production of crops. Although the molecular mechanism for chilling sensitivity has long been debated, no definitive conclusion has so far been reached about its nature. A probable hypothesis, however, is that chilling injury is initiated by phase transition of lipids of cellular membranes, as demonstrated for cyanobacteria, which serve as a model system for the plant cells. Because the phase transition temperature depends on the degree of unsaturation of fatty acids of the membrane lipids, it is predicted that the chilling tolerance of plants can be altered by genetically manipulating fatty-acid desaturation by introducing double bonds into fatty acids of membrane lipids. Here we report the cloning of a gene for the plant-type desaturation (termed desA). The introduction of this gene from a chilling-resistant cyanobacterium, Synechocystis PCC6803, into a chilling-sensitive cyanobacterium, Anacystis nidulans, increases the tolerance of the recipient to low temperature.

Amino Acid Sequence↗
PubMed1989

Chromophore conformational analysis in phycocyanin and in related chromopeptides by surface enhanced Raman spectroscopy.

Chromopeptides got from phycocyanin by proteolytic digestion do not preserve the extended chromophore conformations characteristic to the native protein. Chromophore conformations in the chromopeptides showed heterogenity varying between completely folded and semi-extended states. Indications were found that the silver sol-phycocyanin interaction involves the UV electronic transition of the biliprotein which may explain why the visible excited surface enhanced Raman spectra were similar not to the visible excited but to the UV-excited resonance Raman spectrum of phycocyanin.

Peptide Fragments↗
PubMed1989

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii.

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K(m) for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H(2) adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).

Journal Article↗
PubMed1988

Lipid Saturation Induced Microviscosity Increase Has No Effect on the Reducibility of Flash-Oxidized Cytochrome f in Pea Thylakoids.

Homogeneous catalytic hydrogenation was used to modify the level of fatty acid unsaturation of thylakoid membranes in the pea chloroplast. Fluidity alteration has been monitored simultaneously using the spin-label probe, 16-doxyl stearate. Even in the case of 30% hydrogenation, no change in the reduction rate of flash-oxidized cytochrome f was observed, in contrast to the fact that the same decrease in the double-bond content of the thylakoid membrane resulted in a pronounced inhibition in the full-chain electron transport. We conclude that the rate of lateral diffusion of reduced plastoquinone is unaffected by the lowering of the fluidity of the thylakoid lipid matrix.

Journal Article↗
PubMed1987

Nitrate starvation induces homeoviscous regulation of lipids in the cell envelope of the blue-green alga, Anacystis nidulans.

Replacement of the normal culture liquid to a nitrate-free medium resulted in an immediate drop in the ratio of protein to lipid in isolated cell envelopes of Anacystis nidulans cells. The relative fluidity of the envelope membranes or liposomes, made from the extracted lipids of the envelope, was estimated by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. A thermotrophic phase transition of lipids within the cytoplasmic membrane of intact cells was also revealed by detecting the temperature-dependent absorption changes in the proportion of zeaxanthin at 390 nm. It became evident that a decrease in the proportion of protein to lipid within the cell envelope was accompanied neither by changes in the microviscosity level, nor by shifting of characteristic temperatures of the liquid-crystalline-to-gel transition of lipids. In parallel with nitrate starvation, however, the proportion of saturated fatty acids of the envelope lipids increased markedly. Accumulation of saturated, longer-chain (C18) fatty acids at the cost of C16 counterparts upon nitrate deprivation occurred in all of the complex lipids. In accordance with these findings, a pronounced decrease in the fluidity was demonstrated for the liposomes prepared from the envelope polar lipids of nitrate-starved cells compared with the corresponding control, throughout the temperature range (45-5 degrees C) studied. We propose that the fluidizing effect due to a fall in the ratio of protein to lipid was compensated by a rapidly triggering regulatory process which enables the preservation of the fluidity characteristics at an optimal level within the cell envelope of A. nidulans.

Cell Membrane↗
PubMed1986

Primary Role of the Cytoplasmic Membrane in Thermal Acclimation Evidenced in Nitrate-Starved Cells of the Blue-Green Alga, Anacystis nidulans.

The lipid phase transition of the cytoplasmic membrane and the chilling susceptibility were studied in nitrate-starved Anacystis nidulans cells. Nitrate starvation resulted in the disappearance of the thylakoid membrane system, without any effect on chilling susceptibility. The chilling susceptibility of the algal cells depended on the growth temperature. Temperatures of lipid phase transitions of the cytoplasmic membranes were detected by chilling-induced spectral changes in the carotenoid region, in vivo. These values were identical to those of cultures containing intact thylakoid systems. Our results suggest that cytoplasmic membrane plays a determinative role in the thermal acclimation of the alga cells.

Journal Article↗
PubMed1985

Resonance Raman spectra of phycocyanin, allophycocyanin and phycobilisomes from blue-green alga Anacystis nidulans.

Resonance Raman spectra of native C-phycocyanin, allophycocyanin and whole, intact phycobilisomes from the blue-green alga Anacystis nidulans (Synechococcus 6301) are reported. A tentative assignment for the more prominent resonance Raman bands is suggested. The possibly sensitive regions for inter-chromophore interactions in the case of phycobilisomes are also discussed.

Cyanobacteria↗