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Biomedical subjects

Rattiyaporn Kanlaya

Publications and source records attributed to Rattiyaporn Kanlaya.

5 recordsLinked to original sources

PubMed2006

Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement.

BACKGROUND: Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS: The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS: Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS: Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.

Adolescent↗

PubMed2006

Quantitative analysis and evaluation of the solubility of hydrophobic proteins recovered from brain, heart and urine using UV-visible spectrophotometry.

There is a need for a simple method that can directly quantify hydrophobic proteins. UV-visible spectrophotometry was applied in the present study for this purpose. Absorbance at lambda = 280 nm (A280) was detected for both Escherichia coli membrane proteins and bovine serum albumin, whereas absorbance at lambda = 620 nm (A620) was only detected for E. coli membrane proteins. The A620 values of the brain samples were greater than those of heart samples when equal concentrations were used, regardless of the type of solubilizing agent employed. Because hydrophobic proteins tend to form colloidal microparticles in solution, we also applied UV-visible spectrophotometry to evaluate the efficacies of different extraction protocols for solubilizing hydrophobic proteins. For brain protein extraction, the highest A620 was observed in samples recovered using Tris, whereas the lowest was from samples recovered using SDS. Solubilizing brain tissue with 0.25% SDS (above the CMC) gave a lower A620 than extraction with 0.025% SDS (below the CMC). Addition of 0.25% SDS to samples recovered with Triton caused A620 to drop. A620 could also be used to distinguish between the hydrophobic fractions (pellets) of brain and urine proteins and their hydrophilic fractions (supernatants) prefractionated using high-speed centrifugation. Additionally, an A620/A280 ratio exceeding 0.12 appears to denote highly hydrophobic samples. Our data suggest that direct UV-visible spectrophotometry can be used as a simple method to quantify and evaluate the solubilities of hydrophobic proteins.

Animals↗

PubMed2006

Proteomic identification of alterations in metabolic enzymes and signaling proteins in hypokalemic nephropathy.

Hypokalemic nephropathy caused by prolonged K(+) deficiency is associated with metabolic alkalosis, polydipsia, polyuria, growth retardation, hypertension, and progressive tubulointerstitial injury. Its pathophysiology, however, remains unclear. We performed gel-based, differential proteomics analysis of kidneys from BALB/c mice fed with high-normal-K(+) (HNK), low-normal-K(+) (LNK), or K(+)-depleted diet for 8 wk (n = 6 in each group). Plasma K(+) levels were 4.62 +/- 0.35, 4.46 +/- 0.23, and 1.51 +/- 0.21 mmol/L for HNK, LNK, and KD mice, respectively (p < 0.0001; KD vs. others). With comparable amounts of food intake, the KD mice drank significantly more water than the other two groups and had polyuria. Additionally, the KD mice had growth retardation, metabolic alkalosis, markedly enlarged kidneys, renal tubular dilation, intratubular deposition of amorphous and laminated hyaline materials, and tubular atrophy. A total of 33 renal proteins were differentially expressed between the KD mice and others, whereas only eight proteins were differentially expressed between the HNK and LNK groups, as determined by quantitative intensity analysis and ANOVA with Tukey's post hoc multiple comparisons. Using MALDI-MS and/or quadrupole-TOF MS/MS, 30 altered proteins induced by K(+)-depletion were identified as metabolic enzymes (e.g., carbonic anhydrase II, aldose reductase, glutathione S-transferase GT41A, etc.), signaling proteins (14-3-3 epsilon, 14-3-3 zeta, and cofilin 1), and cytoskeletal proteins (gamma-actin and tropomyosin). Some of these altered proteins, particularly metabolic enzymes and signaling proteins, have been demonstrated to be involved in metabolic alkalosis, polyuria, and renal tubular injury. Our findings may lead to a new road map for research on hypokalemic nephropathy and to better understanding of the pathophysiology of this medical disease when the functional and physiological significances of these altered proteins are defined.

Animals↗

PubMed2006

Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and variability.

We performed systematic evaluation of 38 protocols to concentrate normal human urinary proteins prior to 2D-PAGE analysis. Recovery yield and pattern of resolved protein spots were compared among different methods and intra-/inter-individual variabilities were examined. Precipitation with 90% ethanol provided the greatest protein recovery yield (92.99%), whereas precipitation with 10% acetic acid had the least protein recovery (1.91%). In most of precipitation protocols, the higher percentage of applied organic compounds provided the greater recovery yield. With a fixed concentration at 75%, the urine precipitated with acetonitrile had the greatest number of protein spots visualized in 2D gel, whereas the acetic-precipitated sample had the smallest number of spots. For the intra-individual variability, the first morning urine had the greatest amount of total protein but provided the smallest number of protein spots visualized. Excessive water drinking, not caffeine ingestion, caused alterations in the urinary proteome profile with newly presenting spots and also proteins with decreased excretion levels. As expected, there was a considerable degree of inter-individual variability. Coefficients of variation for albumin and transferrin expression were greatest by inter-individual variables. Male urine had greater amount of total protein but provided smaller number of protein spots compared to female urine. These data offer a wealth of useful information for designing a high-quality, large-scale human urine proteome project.

Albumins↗

PubMed2006

Proteomic identification of altered proteins in skeletal muscle during chronic potassium depletion: Implications for hypokalemic myopathy.

Prolonged potassium depletion is a well-known cause of myopathy. The pathophysiology of hypokalemic myopathy, however, remains unclear. We performed a gel-based, differential proteomics study to define altered proteins in skeletal muscles during chronic potassium depletion. BALB/c mice were fed with normal chow (0.36% K+) or K+-depleted (KD) diet (<0.001% K+) for 8 weeks (n = 5 in each group). Left gastrocnemius muscles were surgically removed from each animal. Histopathological examination showed mild-degree infiltration of polymornuclear and mononuclear cells at the interstitium of the KD muscles. Extracted proteins were resolved with two-dimensional electrophoresis (2-DE), and visualized with Coomassie Brilliant Blue R-250 stain. Quantitative intensity analysis revealed 16 up-regulated protein spots in the KD muscles, as compared to the controls. These differentially expressed proteins were subsequently identified by peptide mass fingerprinting and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). Most of the altered proteins induced by chronic potassium depletion were muscle enzymes that play significant roles in several various metabolic pathways. Other up-regulated proteins included myosin-binding protein H, alpha-B Crystallin, and translationally controlled tumor protein (TCTP). These findings may lead to a new roadmap for research on hypokalemic myopathy, to better understanding of the pathophysiology of this medical disease, and to biomarker discovery.

Amino Acid Sequence↗