Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale.

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future.

A comparative study of EGFR mutation screening methods in non-small cell carcinoma of lung.

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase of the ERBB2 family that has important roles in the proliferation and metastasis of tumor cells. It is frequently overexpressed in common solid tumors and has become a favored target for orally administered small-molecule tyrosine kinase inhibitors (TKI) and monoclonal antibody-based therapy. Gain-of-function somatic mutations of the EGFR tyrosine kinase domain have been associated with the response of some patients with non-small-cell lung carcinoma to TKIs. We evaluated three methods of EGFR mutation analysis to identify an optimal assay for clinical testing based on comparison of diagnostic sensitivity, technical difficulty, and cost (Tab. 1, Fig. 1, Ref. 12).

Membrane dynamics as seen by fourier transform infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803. The effects of lipid unsaturation and the protein-to-lipid ratio.

The roles of lipid unsaturation and lipid-protein interactions in maintaining the physiologically required membrane dynamics were investigated in a cyanobacterium strain, Synechocystis PCC 6803. The specific effects of lipid unsaturation on the membrane structure were addressed by the use of desaturase-deficient (desA(-)/desD(-)) mutant cells (which contain only oleic acid as unsaturated fatty acid species) of Synechocystis PCC 6803. The dynamic properties of the membranes were determined from the temperature dependence of the symmetric CH(2) stretching vibration frequency, which is indicative of the lipid fatty acyl chain disorder. It was found that a similar membrane dynamics is maintained at any growth temperature, in both the wild-type and the mutant cell membranes, with the exception of mutant cells grown at the lower physiological temperature limit. It seems that in the physiological temperature range the desaturase system of the cells can modulate the level of lipid desaturation sufficiently to maintain similar membrane dynamics. Below the range of normal growth temperatures, however, the extent of lipid disorder was always higher in the thylakoid than in the cytoplasmic membranes prepared from the same cells. This difference was attributed to the considerable difference in protein-to-lipid ratio in the two kinds of membranes, as determined from the ratio of the intensities of the protein amide I band and the lipid ester C&z.dbnd6;O vibration. The contributions to the membrane dynamics of an ab ovo present 'structural' lipid disorder due to the protein-lipid interactions and of a thermally induced 'dynamic' lipid disorder could be distinguished.

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.

Non-class II HLA gene associated with type 1 diabetes maps to the 240-kb region near HLA-B.

Several studies provide evidence that in addition to the DQ-DR genes, HLA contains another uncharacterized gene or genes associated with type 1 diabetes. Our aim was to investigate the effect of this gene independently of the DQ-DR genes and to localize it with a matched case-control study. More than 1,400 patients and 30,000 control individuals from Finland were studied. They were first genotyped for the selected alleles of the HLA-DQB1, -DQA1, and -DRB1 genes. For the DR3/4(0404) genotype, 75 patients and 181 control subjects were stratified, and 241 patients and 354 controls were stratified for the DR3/4(0401) genotype. Ten microsatellite markers in the HLA class III and I regions (D6S273, TNFa, C12A, STR MICA, MIB, C125, C143, C245, C3211, and MOGc) and selected alleles of the HLA-A and HLA-B genes were studied. In the DR3/4(0404)-stratified group, we found that markers located between C12A and C143 near the HLA-B gene confer a strong additional diabetes association. This was confirmed by the population differentiation test in both DR3/4(0404)- and DR3/4(0401)-stratified groups. Our data indicate that an additional gene associated with type 1 diabetes is located in the 240-kb region near HLA-B. We excluded STR MICA polymorphism as a mutation responsible for diabetes association.

The Unsaturation of Membrane Lipids Stabilizes Photosynthesis against Heat Stress.

The effect of the unsaturation of glycerolipids of thylakoid membranes on the heat tolerance of the photosynthetic evolution of oxygen was studied in vivo by mutation and transformation of fatty-acid desaturases in the cyanobacterium Synechocystis PCC6803. The experimental results indicate that elimination of dienoic lipid molecules decreases, to a small but distinct extent, the heat tolerance of photosynthetic oxygen evolution, but that elimination of trienoic lipid molecules has no effect on the heat tolerance. This conclusion contrasts with the previous hypothesis that the heat tolerance of photosynthesis is enhanced upon an increase in the level of saturation of membrane lipids. It is also shown that light does not affect the nature of the effect of lipid unsaturation on the heat tolerance of photosynthesis.

Genetic Enhancement of the Ability to Tolerate Photoinhibition by Introduction of Unsaturated Bonds into Membrane Glycerolipids.

Strong light leads to damage to photosynthetic machinery, particularly at low temperatures, and the main site of the damage is the D1 protein of the photosystem II (PSII) complex. Here we describe that transformation of Synechococcus sp. PCC 7942 with the desA gene for a [delta]12 desaturase increased unsaturation of membrane lipids and enhanced tolerance to strong light. To our knowledge, this is the first report of the successful genetic enhancement of tolerance to strong light. Analysis of the light-induced inactivation and of the subsequent recovery of the activity of the PSII complex revealed that the recovery process was markedly accelerated by the genetic transformation. Labeling experiments with [35S]L-methionine also revealed that the synthesis of the D1 protein de novo at low temperature, which was a prerequisite for the restoration of the PSII complex, was much faster in the transformed cells than in the wild-type cells. These findings demonstrate that the ability of membrane lipids to desaturate fatty acids is important for the photosynthetic organisms to tolerate strong light, by accelerating the synthesis of the D1 protein de novo.

Review article: transcription factors and growth factors in ulcer healing.

This review is focused on recent investigations demonstrating a pharmacological and pathophysiologic role in gastroduodenal ulceration for growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), as well as for transcription factors. Our experiments revealed accelerated healing, without decreased gastric acid secretion, of chronic cysteamine-induced duodenal ulcers in rats treated daily for 3 weeks with intragastric administration of bFGF, PDGF or VEGF. Our recent studies also indicate a pathophysiological role of endogenous growth factors in the natural history of experimental duodenal ulcer development and healing. More recently, we investigated the genetic regulation of these growth factors in experimental duodenal ulceration. Since gene expression is most effectively controlled by transcription factors, proteins that bind to cis-acting elements of DNA and guide the binding of polymerase II to start the transcription of specific mRNA, we tested the hypothesis that the expression of IEGs and their transcription factor products, such as Egr-1 and Sp1, might precede the increased synthesis of bFGF, PDGF and VEGF in duodenal ulcer healing. Indeed, the duodenal ulcerogen cysteamine, but not its nonulcerogen and toxic analogue ethanolamine, rapidly increased duodenal (but not gastric) mucosal levels of ET-1, which was followed by enhanced expression of Egr-1 and a decrease in Sp1 in the preulcerogenic stage of duodenal ulceration. These changes in levels of ET-1 and expression of transcription factors were also accompanied by increased expression of the CDK inhibitor p21. Thus, not only growth factors such as bFGF, PDGF and VEGF, but also transcription factors such as Egr-1 and Sp1 and the cell cycle regulator p21, may play a role in the natural history of experimental duodenal ulceration.

Gene expression and gene therapy in experimental duodenal ulceration.

Gastroduodenal ulceration is still poorly understood and changes in gene expression may provide new mechanistic insights. Previously, we demonstrated that angiogenic growth factors are potent ulcer healing agents, and the synthesis of bFGF, PDGF and VEGF is enhanced early in duodenal ulcer healing. The initial molecular event in duodenal ulceration seems to be the organ-specific early release of ET-1 in the pre-ulcerogenic stages after the administration of duodenal ulcerogen cysteamine in rats. We also briefly review here data from literature indicating a central role of ET-1 in gastroduodenal ulceration. After studying the involvement of immediate early genes (e.g. egr-1, Sp1) in ulcer development, we now investigated expression of other genes in the duodenal mucosa in the early stages of chemically induced duodenal ulceration in rats. Following a brief review of principles of gene expression and gene therapy, we review our preliminary gene expression studies, involving monitoring about 1200 genes which revealed about 160 signals and prominent changes in about 30 genes in the early stages of experimental duodenal ulceration. Cysteamine enhanced ET-B receptor gene expression in 30 min, while transcription factors (MAX, STAT 3) showed increased expression in 12 h. We recently also initiated gene therapy studies to enhance the local synthesis of PDGF and VEGF to accelerate duodenal ulcer healing, using a single dose of naked DNA (ND) or adenoviral (AV) vectors of VEGF and PDGF in rats with cysteamine-induced duodenal ulcers. Gene therapy with ND or AV of VEGF or PDGF significantly accelerated chronic duodenal ulcer healing, and increased levels of VEGF and PDGF were detected by Western blotting and ELISA in duodenal mucosa after both VEGF and PDGF gene therapy. Thus, gene expression studies provide new insights into the molecular mechanisms of duodenal ulceration and VEGF or PDGF gene therapy seems to be a new option to achieve a rapid ulcer healing.

Growth factors in gastrointestinal diseases.

This review focuses on the recent investigations demonstrating a pharmacological and pathophysiological role for basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) in ulcerative and inflammatory lesions in the upper and lower gastrointestinal (GI) tract. Our initial experiments revealed that intragastric administration of bFGF-w, acid-resistant bFGF-CS23 and PDGF-BB healed chronic cysteamine (mercaptamine)-induced duodenal ulcer in rats, without decreasing gastric acid secretion or concentration. Subsequently we and others have demonstrated that these peptides accelerate the healing of chronic gastric ulcers, chronic erosive gastritis and ulcerative colitis although they have no or modest acute gastric protective activity. Our recent results revealed a decreased bioactivity of bFGF and PDGF in the presence of certain strains of Helicobacter pylori, and this might explain, at least in part, the poor rates of ulcer healing in H. pylori-positive patients. VEGF, in addition to stimulating angiogenesis and granulation tissue production in duodenal ulcer healing, also has an acute gastroprotective effect. New biochemical, molecular biological and immunohistochemical studies indicate that bFGF, PDGF and VEGF play a pathophysiological role in the natural history of ulcer healing. Thus, growth factor research, especially regarding their possible use as a therapeutic tool in duodenal ulcer and colitis, is challenging. On the other hand, in some GI malignancies the diagnostic use of bFGF might be of clinical benefit. However, much research work is needed to transform these 'endogenous drugs' to 'diagnostic tools' and 'exogenous drugs'.

Long-term enhancement of entorhinal-dentate evoked potentials following 'modified' ECS in the rat.

Electroconvulsive therapy (ECT) is widely used as a treatment for drug-resistant depression. The animal analogue of ECT is electroconvulsive shock (ECS) seizures. We have recently shown that repeated ECS seizures cause a long-lasting, perhaps permanent, enhancement in entorhinal-dentate evoked potentials in the rat. Our study, however, involved 'unmodified' ECS, whereas in clinical practice ECT is now usually given in its 'modified' form (with near-threshold currents, a short-acting barbiturate, muscle relaxant and oxygen). We have therefore repeated our experiments using modified ECS. Entorhinal-dentate evoked potentials were measured in Long-Evans rats before and after: (1) eight modified ECS seizures; or (2) eight sham modified ECS trials. Despite the use of the modified procedure, a significant and long-lasting enhancement in population spike amplitude was seen in the ECS group. We conclude that the modified procedure does not protect rats against the long-lasting enhancement of evoked potentials. Similar changes may be occurring in the brains of patients subjected to modified ECT.

Amygdala-kindled and electroconvulsive seizures alter hippocampal expression of the m1 and m3 muscarinic cholinergic receptor genes.

Expression of m1 and m3 muscarinic cholinergic receptors mRNAs was examined in rat hippocampus following either: (1) kindling to five Stage 5 amygdala-kindled seizures; or (2) eight electroconvulsive shock (ECS) seizures. Twenty-four hours after the last seizure of either type, there was a significant decrease in both m1 and m3 mRNAs in CA1, CA3 and the dentate gyrus subfields of the hippocampus. Twenty-eight days after the last seizure of either type, there was a significant increase in m1 mRNAs in CA1, CA3, and the dentate gyrus; for m3 mRNAs, there was a significant increase in CA3 28 days after the last ECS seizure, and in CA1 and CA3 28 days after the last kindled seizure. These results suggest that seizures alter the cholinergic system in the hippocampus, and that some of the alterations are very long-lasting.

Mossy fiber sprouting induced by repeated electroconvulsive shock seizures.

The elicitation of repeated focal seizures (kindling) induces mossy fiber sprouting in the hippocampus of the rat. The present study investigated whether repeated generalized seizures also induce mossy fiber sprouting. Human psychiatric patients receive repeated generalized seizures during electroconvulsive therapy (ECT). Male Long-Evans rats received a course of eight electroconvulsive shock (ECS) seizures administered on a 48-h schedule over a course of 2 1/2 weeks. Control subjects received matched handling, but no stimulation. Fourteen days after the last ECS trial, all subjects were sacrificed and their brains subjected to Timm staining. Cell counts and area measures were also taken in the hilus. Significant sprouting, but not significant cell loss, was seen in the fascia dentata of the subjects that had received ECS.

Ketamine and phenobarbital do not reduce the evoked-potential enhancement induced by electroconvulsive shock seizures in the rat.

Electroconvulsive shock (ECS) seizures provide an animal analog of electroconvulsive therapy (ECT). Repeated ECS seizures cause a long-lasting, and perhaps permanent, enhancement of entorhinal-dentate evoked potentials (EPs) in the rat. Recently it has been reported that ketamine protects against ECS-induced EP enhancement. The present study was designed to replicate these findings and to extend them by incorporating a phenobarbital group (to control for ketamine's partial diminution of seizures) and an animal test of antidepressant activity (the Porsolt test). Unexpectedly, we found that neither ketamine nor phenobarbital protected against ECS-induced enhancement of EPs. Both, however, diminished the 'therapeutic' effects of ECS, as modeled by the Porsolt test. These data suggest that the use of ketamine would not eliminate the unwanted effects of ECT and that it might diminish ECT's therapeutic benefits.

Induction of polyunsaturated fatty-acid synthesis enhances tolerance of a cyanobacterium, Cylindrospermopsis raciborskii, to low-temperature photoinhibition.

Acyl-lipid desaturation introduces double bonds (unsaturated bonds) at specifically defined positions of fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. Desaturation pattern of the glycerolipids of Cylindrospermopsis raciborskii (C. raciborskii), a filamentous cyanobacterial strain, was determined in cells grown at 35 degrees C and 25 degrees C. The lowering of the growth temperature from 35 degrees C to 25 degrees C resulted in a considerable accumulation of polyunsaturated octadecanoic fatty acids in all lipid classes. Lipid unsaturation of C. raciborskii was also compared to Synechocystis PCC6803. In C. raciborskii cells, a shift in growth temperature induced a much more pronounced alteration in the desaturation pattern of all lipid classes than in Synechocystis PCC6803. The tolerance to low-temperature photoinhibition of the C. raciborskii cells grown at 25 degrees C and 35 degrees C was also compared to the tolerance of Synechocystis cells grown at the same temperatures. Lower growth temperature increased the tolerance of C. raciborskii cells but not that of Synechocystis cells. These results strengthen the importance of polyunsaturated glycerolipids in the tolerance to environmental stresses and may give a physiological explanation for the determinative role of C. raciborskii strain in algal blooming in the Lake Balaton (Hungary).

Analysis of extended human leukocyte antigen haplotype association with Addison's disease in three populations.

OBJECTIVE: Addison's disease is an organ-specific autoimmune disorder with a polygenic background. The aim of the study was to identify non-class II human leukocyte antigen (HLA) susceptibility genes for Addison's disease. DESIGN AND METHODS: Addison's disease patients from three European populations were analysed for selected HLA-DR-DQ alleles and for 11 microsatellite markers covering approximately 4 Mb over the HLA region. Subjects were 69 patients with Addison's disease from Estonia (24), Finland (14) and Russia (31). Consecutively recruited healthy newborns from the same geographical regions were used as controls (269 Estonian, 1000 Finnish and 413 Russian). Association measures for HLA-DRB1, DQB1, DQA1 and 11 microsatellites between D6S273 and D6S2223 were taken. A low-resolution full-house typing was used for HLA class II genes, while microsatellite markers were studied using fluorescence-based DNA fragment sizing technology. RESULTS: We confirmed that the HLA-DR3-DQ2 and the DQB1*0302-DRB1*0404 haplotypes confer disease susceptibility. In Russian patients, we also found an increase of DRB1*0403 allele, combined with DQB1*0305 allele in three out of six cases (P<0.0001). Analysis of 11 microsatellite markers including STR MICA confirmed the strong linkage in DR3-DQ2 haplotypes but DRB1*0404-DQB1*0302 haplotypes were diverse. MICA5.1 allele was found in 22 out of 24 Estonian patients, but results from Finnish and Russian patients did not support its independent role in disease susceptibility. CONCLUSION: HLA-DRB1*0403 was identified as a novel susceptibility allele for Addison's disease. Additionally, we found no evidence of a non-class II HLA disease susceptibility locus; however, the HLA-DR3-DQ2 haplotype appeared more conserved in patient groups with high DR-DQ2 frequencies.

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.

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.

Mn and Co toxicity in chlorophyll biosynthesis.

The metal ion-induced inhibition of tetrapyrrole biosynthesis was studied in the cyanobacterium Anacystis nidulans. The accumulation of protoporphyrin and Mg protoporphyrin due to the effect of Co(2+) and Mn(2+) treatment, respectively, pointed to two different sites of inhibition.

Two simple procedures for isolation of allophycocyanin II from Anacystis nidulans.

Allophycocyanin II purification using initial extraction of phycobiliproteins by acetone treatment is introduced. An additional fast method using Al2O3 is described. Both extraction procedures are followed up by conventional hydroxylapatite chromatography.