Refining Transcriptome Gene Catalogs by MS-Validation of Expressed Proteins.

Protein sequence identification by tandem mass spectroscopy (LC-MS/MS) identifies thousands of protein sequences even in complex mixtures, and provides valuable insight into the biological functions of different cells. For non-model organisms, transcriptomes are generally used to allow peptide identification, an important addition to their use as a gene catalog allowing the potential metabolic activities of cells to be determined. We used LC-MS/MS data to identify which of the six possible reading frames in the transcriptome was actually used by the cell to make protein, and asked whether this would have an impact on downstream analyses using the dataset. We combined results from several LC-MS/MS experiments designed to identify peptide sequences in extracts from the dinoflagellate Lingulodinium polyedra using a 74 655-sequence transcriptome. We compiled a list of 6628 translated nucleic acid sequences that contained the ensemble of peptide matches (termed MS-validated sequences) and assessed the similarity in downstream analyses between this data set and the 6628 nucleic acid sequences from which they were derived. When compared with BLASTx analyses of the DNA sequences, the MS-validated protein-sequences-analyzed using BLASTp showed differences in gene ontology, had more identified BLAST hits, and contained more KEGG pathway enzymes. The MS-validated protein sequences also differ from datasets containing longest open reading frame (ORF) protein sequences. We also note a poor correlation between the levels of protein and mRNA abundance, a comparison not previously performed for dinoflagellates. The differences observed between analyses of MS-validated protein sequence and nucleic acid sequence datasets suggest use of the former may provide a more accurate representation of cellular capacity than the latter. Developing MS-validated protein sequence datasets may also speed interpretation of MS-MS spectra in bottom up proteomics experiments.

Label-free MS/MS analyses of the dinoflagellate Lingulodinium identifies rhythmic proteins facilitating adaptation to a diurnal LD cycle.

Protein levels were assessed in the dinoflagellate Lingulodinium polyedra over the course of a diurnal cycle using a label-free LC-MS/MS approach. Roughly 1700 proteins were quantitated in a triplicate dataset over a daily period, and 13 were found to show significant rhythmic changes. Included among the proteins found to be most abundant at night were the two bioluminescence proteins, luciferase and luciferin binding protein, as well as a proliferating cell nuclear protein involved in the nightly DNA replication. Aconitase and a pyrophosphate fructose-6-phosphate-1-phosphotransferase were also found to be more abundant at night, suggestive of an increased ability to generate ATP by glucose catabolism when photosynthesis does not occur. Among the proteins more abundant during the day were found a 2-epi-5-epi-valiolone synthase, potentially involved in synthesis of mycosporin-like amino acids that can act as a "microbial sunscreen", and an enzyme synthesizing vitamin B6 which is known to protect against oxidative stress. A lactate oxidoreductase was also found to be more abundant during the day, perhaps to counteract the pH changes due to carbon fixation by facilitating conversion of pyruvate to lactate. This unbiased proteomic approach reveals novel insights into the daily metabolic changes of this dinoflagellate. Furthermore, the observation that only a limited number of proteins vary support a model where metabolic flux through pathways can be controlled by variations in a select few, possibly rate limiting, steps. Data are available via ProteomeXchange with identifier PXD006994.

Exploring dinoflagellate biology with high-throughput proteomics.

Dinoflagellates are notorious for their ability to form the harmful algal blooms known as "red tides," yet the mechanisms underlying bloom formation remain poorly understood. Despite recent advances in nucleic acid sequencing, which have generated transcriptomes from a wide range of species exposed to a variety of different conditions, measuring changes in RNA levels have not generally produced great insight into dinoflagellate cell biology or environmental physiology, nor do we have a thorough grasp on the molecular events underpinning bloom formation. Not only is the transcriptomic response of dinoflagellates to environmental change generally muted, but there is a markedly low degree of congruency between mRNA expression and protein expression in dinoflagellates. Herein we discuss the application of high-throughput proteomics to the study of dinoflagellate biology. By profiling the cellular protein complement (the proteome) instead of mRNA (the transcriptome), the biomolecular events that underlie the changes of phenotypes can be more readily evaluated, as proteins directly determine the structure and the function of the cell. Recent advances in proteomics have seen this technique become a high-throughput method that is now able to provide a perspective different from the more commonly employed nucleic acid sequencing. We suggest that the time is ripe to exploit these new technologies in addressing the many mysteries of dinoflagellate biology, such as how the symbiotic dinoflagellate inhabiting reef corals acclimate to increases in temperature, as well as how harmful algal blooms are initiated at the sub-cellular level. Furthermore, as dinoflagellates are not the only eukaryotes that demonstrate muted transcriptional responses, the techniques addressed within this review are amenable to a wide array of organisms.

The time effect of pressure on tissue viability: investigation using an experimental rat model.

An experimental rat model was used to investigate the time-pressure effect on tissue viability. External loading equivalent to 13.3 kPa (100 mm Hg) of pressure was applied to the greater trochanter and tibialis area of Sprague-Dawley rats using pneumatic indentors for duration of 6 hrs each day for 1 to 4 days. It was observed that postocclusive hyperemic responses were gradually increased at the trochanter throughout the 4 days of loading, whereas for the tibia there was a significant increase (P = 0.04) in postocclusive hyperemic flow between Days 2 and 3. In histologic evaluations, cutaneous tissue damage was observed at the trochanter area but not at the tibialis area after 2 consecutive days of load application. In contrast, degeneration of muscle cells characterized by numerous increases of nuclei occupying the central of the muscle fibers was observed after 2 days of load application at the tibialis. The situation was found to progress with time (P = 0.17). The presence of other histologic signs, including the internalization of peripherally located nuclei, replacement of muscle cells by fibrosis and adipose tissues, and the presence of pyknotic nuclei as well as karyorrhexis, confirmed that the affected tissues were damaged. These findings suggest that postocclusive hyperemia and the distress of tissues under loading could be closely related.

Comparative proteomic studies of a Scrippsiella acuminata bloom with its laboratory-grown culture using a 15N-metabolic labeling approach.

Comparative proteomic analysis was carried out using cells isolated from a natural bloom of Scrippsiella acuminata (formerly Scrippsiella trochoidea) in the early bloom (EB) and late bloom (LB) stages as well as with laboratory-grown cultures of cells isolated from the bloom in early growth (EG) and late growth (LG) stages. For quantitative proteomics, LG cells were grown for 20 generations in the presence of 15N as a reference (i.e. common denominator) for all comparison. In comparisons with early growth laboratory grown cells (EG/LG), nearly 64% of proteins identified had similar abundance levels, with the remaining 36% mostly more abundant in EG cells. Calvin cycle, amino acid metabolism, chlorophyll biosynthesis and transcription/translation were among the up-regulated processes. Cells from the early bloom (EB/LG) had a greater abundance of transporters and enzymes related to light harvesting and oxidative phosphorylation, while the abundance of these proteins decreased in late bloom cells (LB/LG). All natural bloom samples showed either constant or lower abundance levels of enzymes involved in sugar synthesis and glycolytic pathways compared to laboratory grown cells. Our results represent the first examination of the proteomic changes in the development of a natural dinoflagellate bloom. Importantly, our results demonstrate that the proteome of cells grown in the laboratory is distinctively different from cells in a natural bloom.

Effects of prolonged surface pressure on the skin blood flowmotions in anaesthetized rats--an assessment by spectral analysis of laser Doppler flowmetry signals.

The objective of this study is to assess the effect of prolonged surface compression on the skin blood flowmotion in rats using spectral analysis based on wavelets transform of the periodic oscillations of the cutaneous laser Doppler flowmetry (LDF) signal. An external pressure of 13.3 kPa (100 mmHg) was applied to the trochanter area and the distal lateral tibia of Sprague-Dawley rats via two specifically designed pneumatic indentors. The loading duration was 6 hours/day for 4 consecutive days. Five frequency intervals were identified (0.01-0.04 Hz, 0.04-0.15 Hz, 0.15-0.4 Hz, 0.4-2 Hz and 2-5 Hz) corresponding to endothelial related metabolic, neurogenic, myogenic, respiratory and cardiac origins. The absolute amplitude of oscillations of each particular frequency interval and the normalized amplitude were calculated for quantitative assessments. The results showed that (1) tissue compression following the above schedule induced significant decrease in the normalized amplitude in the frequency interval of 0.01-0.04 Hz both in the trochanter area (p < 0.001) and tibialis area (p = 0.023), (2) prolonged compression induced significant increase in the absolute amplitude (p = 0.004 for the trochanter area and p = 0.017 for the tibialis area) but significant decrease in the normalized amplitude (p = 0.023 for the trochanter area and p = 0.026 for the tibialis area) in the frequency interval of 0.15-0.4 Hz, and (3) at the tibialis area, the flowmotion amplitude (frequency interval 0.15-0.4 Hz) measured prior to the daily tissue compression schedule was found to be significantly higher on day 4 than the measurements obtained on day 1. However, this finding was not observed at the trochanter area. Our results suggested that prolonged compression might induce endothelial damage and affect the endothelial related metabolic activities.

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor.

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.

Electro-acupuncture potentiates the disulphide-reducing activities of thioredoxin system by increasing thioredoxin expression in ischemia-reperfused rat brains.

Reactive oxygen species can directly affect the conformation and activity of sulfhydryl-containing proteins by oxidation of their thiol moiety. During the process of ischemia-reperfusion, the thioredoxin (Trx) system (consisting of thioredoxin reductase (TR), Trx and NADPH) prevents susceptible proteins from this oxidative modification. Oxidative damage is one of the most damaging stress in ischemia. If oxidative stress could be minimized, the damage occurred will be minimized accordingly. We therefore investigated whether electroacupuncture (EA) treatment at Fengchi (GB20) or Zusanli (ST36) acupoints in post-ischemic rats could increase TR-related activities and Trx expression which would translate into maintaining the intact thiol moiety of susceptible proteins in the surrounding. Our results indicated that EA treatment at either acupoint increased the Trx expression in ischemic-reperfused brain tissues. Induced Trx expressed levels gradually increased from post-ischemia day 1 to day 4. Statistical analysis revealed that there was no observable difference in the effect of EA treatment at GB20 and ST36. Sham EA treatment did not induce any Trx expression. EA at either acupoint did not alter TR activities in both non-ischemic and ischemic-reperfused rat brains. Taken overall, our finding suggests that EA treatment at GB20 or ST36 could increase Trx expression which could minimize oxidative modifications of thiol groups of surrounding proteins.

Electroacupuncture reduces the extent of lipid peroxidation by increasing superoxide dismutase and glutathione peroxidase activities in ischemic-reperfused rat brains.

Reactive oxygen species can be scavenged by superoxide dismutase (SOD) and glutathione peroxidase (GPx). During ischemia-reperfusion, the normal functioning of these antioxidant enzymes may be insufficient for the prevention of oxidant-induced peroxidation of membrane lipids and hence cerebral infarction. We therefore investigated whether electroacupuncture (EA) treatment at Fengchi points in post-ischemic rats could increase the antioxidant enzyme activities and thereby reduce the extent of lipid peroxidation. The results indicated that while EA did not alter the antioxidant enzyme activities in non-ischemic normal rat brains, ischemia-reperfusion caused significant increases in SOD and GPx activities. EA treatment further increased the antioxidant enzyme activities in ischemic-reperfused brain tissues, with a concomitant decrease in the extent of lipid peroxidation. Our finding suggests that EA treatment at Fengchi reduced the extent of lipid peroxidation in ischemic-reperfused rat brains, possibly by increasing the activities of SOD and GPx.

Effectiveness of multiple pre-ischemia electro-acupuncture on attenuating lipid peroxidation induced by cerebral ischemia in adult rats.

Free radicals induced by cerebral ischemia-reperfusion injury can trigger lipid peroxidation, leading to the production of malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE). Post-ischemia electroacupuncture (EA) therapy was able to reduce extent of lipid peroxidation. However, the effect of pre-ischemic EA therapy has not been reported. In this study, we aim to investigate the effectiveness of pre-ischemic EA therapy on lipid peroxidation in the rat ischemic injury model. Four groups of Sprague-Dawley rats were designed: Placebo group (without EA therapy), NA group (EA therapy on non-acupoint), GB20 group (EA therapy on Fengchi), and ST36 group (EA therapy on Zusanli). Half of each group (n = 6) received 30-minute EA therapy for 3 times and the other half group for 18 times before the occlusion of right middle cerebral artery. Right brains were taken for determination of concentration of MDA and the total of MDA plus 4-HNE. We found that multiple pre-ischemia EA therapy at either GB20 or ST36 can effectively reduce the amount of MDA produced after MCA occlusion. However, this reduction was not observed in the total amount of MDA and 4-HNE. In conclusion, pre-ischemia EA can partly regulate the lipid peroxidation in cerebral ischemia, where both GB20 and ST36 have a similar beneficial effectiveness.

Application of fluorescence difference gel electrophoresis technology in searching for protein biomarkers in chick myopia.

The lens-induced myopia (LIM) in response to concave lens (negative lens) is a well established animal model for studying myopia development. However, the exact visual and neurochemical signaling mechanisms involving myopic eye growth are yet to be elucidated. The feasibility of applying a novel two-dimensional fluorescence difference gel electrophoresis technique for global protein profilings and a search for differential protein expressions in LIM were explored in the present study. Two-dimensional polyacrylamide gel electrophoresis was performed employing a "minimal Lysine labeling" approach and a reverse CyeDye experimental protocol using retinal tissue from chicks. The retinal protein profiles between myopic and control eyes were found to be very similar. More than a thousand protein spots could be detected on a 2D gel. Sixteen and ten protein spots were found to be up-regulated and down-regulated respectively in the myopic eyes according to our preset criteria with the inclusion of an internal pool standard. About 65% of those filtered spots could be successfully identified by peptide mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry . Most of the differentially expressed proteins were found to be related to cytoskeletal or oxidative functions. According to the prediction of subcellular locations, most of them (about 84%) were classified as cytoplasmic proteins. The cellular functions for those differentially expressed proteins were reported and their possible involvements in the compensated eye growth were discussed. We have optimized a workable protocol for the study of the differential retinal protein expressions in the LIM using 2D-DIGE approach which was shown to have a number of advantages over the traditional 2D electrophoresis technique.

A chick retinal proteome database and differential retinal protein expressions during early ocular development.

Proteomics approach as a research tool has gained popularity in a growing number of basic and clinical researches. However, proteomic research has yet to gain significant momentum in eye research. Hence, we decided to build a retinal proteome database using postnatal retinal tissue from chick, a commonly used animal model in eye research. Employing 2-D gels with the coverage of 3-10 pH gradients, we were able to resolve hundreds of proteins from young chick retinae. Among them, 155 high abundant proteins were identified by Peptide Mass Fingerprinting (PMF) after the Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS). These proteins were then classified according to their functions. Making use of the retinal database, we were able to identify several differentially expressed proteins that might be involved in early retinal development by comparing the 2-DE maps of chick retinal tissues (3, 10, and 20 days after hatching). With the current proteomics approach, we not only documented the most abundant soluble proteins in the chick retinal tissue, but also demonstrated the dynamic protein expression changes during early ocular development. This represents one of the first steps in building a complete protein database in chick retinae which is applicable to the study of eye diseases from a few selected protein candidates to the whole proteome. Proteomic technology may provide a high throughput platform for advancing eye research in the feasible future.

Cloning and characterization of Arabidopsis thaliana pyridoxal kinase.

Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.

Treatment of experimentally induced transient cerebral ischemia with low energy laser inhibits nitric oxide synthase activity and up-regulates the expression of transforming growth factor-beta 1.

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) has been shown to be neurotoxic while transforming growth factor-beta 1 (TGF-beta1) is neuroprotective in the stroke model. The present study investigates the effects of low energy laser on nitric oxide synthase (NOS) and TGF-beta1 activities after cerebral ischemia and reperfusion injury. STUDY DESIGN/MATERIALS AND METHODS: Cerebral ischemia was induced for 1 hour in male adult Sprague-Dawley (S.D.) rats with unilateral occlusion of middle cerebral artery (MCAO). Low energy laser irradiation was then applied to the cerebrum at different durations (1, 5, or 10 minutes). The activity of NOS and the expression of TGF-beta1 were evaluated in groups with different durations of laser irradiation. RESULTS: After ischemia, the activity of NOS was gradually increased from day 3, became significantly higher from day 4 to 6 (P < 0.001), but returned to the normal level after day 7. The activity and expression of the three isoforms of NOS were significantly suppressed (P < 0.001) to different extents after laser irradiation. In addition, laser irradiation was shown to trigger the expression of TGF-beta1 (P < 0.001). CONCLUSIONS: Low energy laser could suppress the activity of NOS and up-regulate the expression of TGF-beta1 after stroke in rats.

Conformational changes in the reaction of pyridoxal kinase.

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK),1 we determined the crystal structures of PLK.AMP-PCP-pyridoxamine, PLK.ADP.PLP, and PLK.ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK.ATP complex. The distance between the reacting groups of the two substrates is 5.8 A apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK.ADP.PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B(6). Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized.

Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamily.

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.