Prolactin and upstream migration of the amphidromous teleost, ayu Plecoglossus altivelis.

Changes in mRNA levels of prolactin (PRL) during the upstream migration were examined in fry of the amphidromous fish, ayu Plecoglossus altivelis. Quantification of mRNA has been done with real-time PCR and expressed as whole body or pituitary contents depending the body size of fry. PRL mRNA levels of ayu caught in seawater of the coastal area remained low during early spring. Prior to the start of the upstream migration, the fish caught in the coastal area in mid spring showed increased levels of PRL mRNA. There were further increases in PRL levels in the fish caught in the river. Analysis of proportions revealed that there were significant differences among PRL mRNA in the fish caught in different environmental salinities. Body weight showed a positive relation with PRL mRNA in ayu caught in seawater. A landlocked population of ayu, which migrates from lake to river, showed no significant change in PRL mRNA levels before and after upstream migration. Results in this study indicate the importance of up-regulation of PRL gene expression of ayu during the upstream migration from seawater to fresh water. There is a possible relationship between body size and PRL in the early developmental stage of ayu in seawater, but not in the fish in fresh water.

Changes in prolactin mRNA levels during downstream migration of the amphidromous teleost, ayu Plecoglossus altivelis.

Changes in mRNA levels of prolactin (PRL) during seaward migration and after experimental transfer from fresh water (FW) to seawater (SW) were examined in larvae of the amphidromous fish, ayu Plecoglossus altivelis. In the field study, ayu larvae caught in the surf zone showed lower levels of PRL mRNA than those in the river, while growth hormone (GH) levels showed no significant change. Decrease in PRL gene transcription was also observed 24h after direct transfer from FW to SW, whereas there was no significant influence of water temperature. On the other hand, there was no significant change in GH mRNA levels in relation to SW transfer or environmental temperature. In a raceway with a vertical salinity gradient, PRL mRNA levels of ayu larvae showed a significant reduction during spontaneous migration from FW to SW, which mimicked the results from the field observation and the transfer experiment, and then a gradual increase during the course of development. Whole body water and sodium contents of larvae in a salinity gradient were stable during migration to SW. Results in this study indicated the importance of regulation of PRL gene expression in the downstream migration and acclimation to SW during the early development of ayu.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUND: Cystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level. METHODS: We used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA. RESULTS: In normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine. CONCLUSION: In the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Low-density lipoprotein (LDL), which includes apolipoprotein A-I (apoAI-LDL) as a novel marker of coronary artery disease.

BACKGROUND: Serum low-density lipoprotein (LDL), which includes apolipoprotein A-I (apoAI-LDL) may be generated by oxidization in the serum of patients with coronary artery disease (CAD). We determine the utility of the serum apoAI-LDL level as a novel coronary risk factor. METHODS: We measured serum apoAI-LDL in 473 consecutive patients who underwent diagnostic coronary angiography. Serum levels of apoAI-LDL were assayed by a newly developed ELISA. RESULTS: The patients consisted of 84 with unstable angina (UA), 259 with stable CAD, and 130 without CAD (control). The serum level of apoAI-LDL was higher in CAD patients than in the control group (31.4 (22.1-41.4) microg/ml vs. 24.6 (18.4-29.2) microg/ml, respectively, p<0.001), as well as in patients with UA compared to those with stable CAD 44.5 (35.8-51.9) microg/ml vs. 27.1 (19.5-35.6) microg/ml, respectively, p<0.0001) (data are expressed as the median (25th-75th percentiles)). By logistic regression analysis, only apoAI-LDL was independent, being significantly able to predict CAD (odds ratio: 1.50, 95% CI: 1.23-1.82, p<0.001), and differentiate unstable angina (odds ratio: 1.80, 95% CI: 1.48-2.17, p<0.001) after controlling for classical risk factors. CONCLUSION: The serum level of apoAI-LDL, a newly identified component of oxidized LDL, may be a more sensitive marker of CAD and acute coronary syndrome than CRP.

Highly selective encaging of carbon dioxide molecules in the mixed carbon dioxide and nitrogen hydrate at low temperatures.

The structural identification and guest compositions of the mixed CO(2) and N(2) hydrates at low temperature conditions were investigated by both theoretical predictions and experimental measurements. From the model calculations, at very low temperatures, the highly CO(2)-concentrated hydrates over 95 mol % CO(2) on the basis of water-free concentration could coexist with the gas mixtures of low CO(2) concentrations in equilibrium. X-ray diffraction measurements of the hydrates formed with the gas mixture of 3.16 mol % CO(2) and balanced N(2) indicate that the formed hydrates at all conditions considered in this study were identified as structure I, whereas the model predicts a structural transition to structure II around 220 K. However, it was also found that the formed hydrate samples contain a considerable amount of hexagonal ice resulting from incomplete conversion of ice to the hydrates. The compositional analysis suggests that a favorable encaging of CO(2) in the mixed hydrate can be obtained by the hydrate formation at low temperatures and relative amount of CO(2) molecules in the mixed hydrates increases with a decrease of temperature.

Valsartan reduces serum cystatin C and the renal vascular resistance in patients with essential hypertension.

A high level of albuminuria and increased renal vascular resistance are associated with hypertensive renal damage. In this study, the authors investigated the effect of the angiotensin II receptor blocker, valsartan, on renal function and intrarenal hemodynamics in non-diabetic patients with essential hypertension. A prospective three-month study of the effects of valsartan, 40-80 mg/day, was performed in 30 hypertensive patients. As an assessment of renal function, serum creatinine, urine albumin/creatinine (Alb/Cr) ratio, and serum cystatin C levels were evaluated. Doppler ultrasonography of the kidney was performed for the evaluation of renal hemodynamics. Peak-systolic, end-diastolic, and mean velocities of interlobar arteries were evaluated, and the pulsatility index (PI) and resistive index (RI) were calculated. It was determined that patients with microalbuminuria had higher levels of serum cystatin C, PI, and RI compared to patients without microalbuminuria. Valsartan treatment significantly reduced systolic and diastolic blood pressure and decreased the Alb/Cr ratio. Serum creatinine was not changed, whereas serum cystatin C levels were significantly reduced. Valsartan treatment significantly decreased the PI in all patients and both PI and RI in patients with microalbuminuria. These results suggest that the angiotensin II receptor blocker, valsartan, is able to improve renal function by reducing renal vascular resistance in hypertensive patients, especially in patients with microalbuminuria, and may prevent future renal failure in patients with essential hypertension.

Identification of specific autoantigens in Sjögren's syndrome by SEREX.

We carried out SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using sera from patients with Sjögren's syndrome (SjS) and investigated the frequencies of autoantibodies against autoantigens identified by SEREX in the sera of healthy individuals (HI) and patients with SjS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). IFI16 and two kelch-like proteins, KLHL12 and KLHL7, were found to be novel autoantigens in SjS by SEREX. A markedly high frequency of anti-IFI16 autoantibodies was observed in the sera of SjS (SjS, 70%; RA, 13%; SLE, 33%; HI, 0%). Interestingly, all serum samples from SjS demonstrated immunoreactivity against one or both of IFI16 and SS-B/La. The presence of autoantibodies against KLHL12 and KLHL7 in the sera was significantly specific to SjS (23% and 17%, respectively), as they were not detected in RA, SLE or HI. Furthermore, we confirmed that transcripts of these autoantigens were expressed preferentially in the salivary glands and immuno-privileged testes. Our results suggest these autoantigens may be useful as serological markers for the clinical diagnosis of SjS and may play a crucial role as organ-specific autoantigens in the aetiopathogenesis of SjS. This study warranted clinical evaluations of autoantibodies against IFI16, KLHL12 and KLHL7 in combination with anti-SS-B/La autoantibodies.

Production and characterization of an active single-chain variable fragment antibody recognizing CD25.

The alpha subunit of the interleukin-2 receptor (IL-2Ralpha, CD25) is a potential target in therapeutic approaches for hematolopoietic malignancies expressing CD25 on their cell surface, such as adult T cell leukemia/lymphomas. Recent reports have demonstrated that depletion of CD4+CD25+ regulatory T cells with anti-CD25 antibodies may enhance host tumor immunity. We previously raised a mouse monoclonal antibody (mAb), Ta60b mAb (IgG1kappa), specifically recognizing CD25, and an attempt was made here to produce a single chain Fv fragment (scFv) from this mAb as an initial step to development of scFv-based therapeutics. cDNA fragments encoding for the variable regions of the light and heavy chains of the Ta60b mAb were thus isolated by polymerase chain reaction-mediated cloning, and, an expression vector constructed to express Ta60b scFv fused with the maltose binding protein (MBP) in the periplasm of Escherichia coli. The soluble form of MBP-Ta60b fused scFv could be extracted and affinity-purified with an amylose/agarose column, allowing its immunoreactivity to be analyzed by enzyme-linked immunosorbent assay (ELISA), mixed hemadsorption assay, and fluorescence activated cell sorting. In addition, binding activity was studied by competitive ELISA and surface plasmon resonance. The results showed that Ta60b scFv obtained from periplasm retains good reactivity, although its KD value was 4-fold lower than that of the whole Ta60b antibody, suggesting possible clinical use for treatment of patients with CD25-expressing tumors and also for enhancing anti-tumor immunity.

A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease.

BACKGROUND: Although some reports have indicated that acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) can predict the prognosis in patients with acute coronary syndrome, the value of these markers in patients with stable coronary artery disease (CAD) still remains obscure. Therefore, our aim was to determine the prognostic value of inflammatory markers in patients with stable coronary artery disease. METHODS AND RESULTS: We conducted a prospective cohort study in 140 consecutive patients with stable coronary artery disease who had at least one coronary stenosis more than 50% in diameter seen on diagnostic coronary angiography (CAG). We determined serum levels of the SAA/LDL complex as a new marker in addition to CRP and SAA. Serum levels of the SAA/LDL complex were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). End-points were defined as cardiac death, myocardial infarction, cerebral infarction, and coronary revascularization. End-point events occurred in 21 patients (2 death from myocardial infarction, 2 cerebral infarction, and 17 revascularization). Age (year) (OR = 1.14, CI: 1.05-1.25), diabetes mellitus (OR = 3.50, CI: 1.08-11.40), triglyceride (10mg/dl) (OR = 1.12, CI: 1.01-1.23) and SAA/LDL complex (10 microg/ml) (OR = 2.32, CI: 1.05-4.70) were independently related to the events. A reconstitution experiment suggested that the SAA/LDL complex is derived by oxidative interaction between SAA and lipoproteins. CONCLUSIONS: The SAA/LDL complex reflects intravascular inflammation directly and can be a new marker more sensitive than CRP or SAA for prediction of prognosis in patients with stable coronary artery disease.

[Development of blood examination method of serum amyloid A and LDL complex, and clinical application to prediction of cardiovascular event].

In recent years, it has been reported that the acute-phase proteins C-reactive protein(CRP) and serum amyloid A(SAA), the sera levels of which are elevated in inflammation, are also elevated in coronary artery disease such as acute myocardial infarction. Also, high-sensitivity CRP assay is thought to be useful in predicting the prognosis of coronary heart disease. While investigating complexes of acute-phase proteins and low-density lipoprotein(LDL), we found a complex of LDL and SAA(SAA/LDL complex). The SAA/LDL complex in blood are formed from LDL and HDL by an oxidation reaction. Therefore, we developed an ELISA using anti-human SAA antibody and anti-human apoB, and determined a new method for measuring SAA/LDL complex in sera. We evaluated SAA/LDL complex as a new marker for prediction of prognosis in addition to the ordinary markers in consecutive 140 patients with stable coronary heart disease who had at least 1 coronary artery stenosis more than 50% in diameter at the diagnostic coronary angiography. Of these 140 patients, 2 developed fatal myocardial infarction, 2 cerebral infarction, and 17 angina pectoris requiring coronary revascularization therapy during 1 year and 6 months after blood examinations. The SAA/LDL complex value in this EVENT group of 21 patients was significantly higher than that in the control group of 119 individuals. High-sensitivity CRP (hs-CRP) assay and SAA measurement showed no significant difference between the 2 groups. The SAA/LDL complex reflects intravascular inflammation directly and can be a new marker more sensitive than hs-CRP or SAA for prediction of prognosis in patients with stable coronary artery disease.

Measurement of cystatin-C and creatinine in urine.

BACKGROUND: The concentration of serum cystatin-C (Cys-C) is highly correlated with creatinine (Cr), and is mainly determined by glomerular filtration; thus, Cys-C may be an index of the glomerular filtration rate (GFR). However, the kinetics of urinary Cys-C and Cr excretions are unclear. Thus, we investigated the kinetics of urinary Cys-C and Cr excretions, and examined whether the urinary Cys-C concentration can be used as a marker of renal function. METHODS: The urinary excretion of Cys-C and Cr was evaluated in 1670 healthy subjects and 217 patients with proteinuria. We also investigated the urinary Cys-C concentration in 52 patients with chronic renal failure. RESULTS: There was a good correlation between the urinary concentrations of Cys-C and Cr in the healthy group. This relation was also observed in patients showing persistent proteinuria without tubular cell damage. The mean urinary Cr concentration increased with age, and it was affected by the muscle mass. In contrast, the urinary Cys-C concentration was not affected by the muscle mass, and the concentration remained constant for all ages. We further found that the ratio of Cys-C to Cr (CCR) is a good index of the state of Cys-C reabsorption in the proximal tubules. CONCLUSIONS: The urinary CCR can be a marker of renal tubular dysfunction. In addition, when CCR was in the normal range, the urinary Cys-C concentration accurately reflected the glomerular filtration function.

Serum cystatin-C values in children by age and their fluctuation during dehydration.

BACKGROUND: Serum creatinine level (Cr) is extensively used as an index of glomerular filtration rate (GFR) with bedside patients. As serum Cr level differ by the muscle volume, the use of serum Cr as an index of GFR has long been considered problematic in children. METHODS: The subjects of this study were 135 healthy children aged from 1 to 15-years-old. As a pathological condition, the changes in serum cystatin C (Cys-C) in the children whose blood urea nitrogen exceeded 20 mg/dL during the acute stage of dehydration were also investigated. RESULTS: The mean serum Cys-C value was 0.67 +/- 0.19 mg/L on the whole. There was not any male-female difference. The mean serum Cys-C values of the 12 cases in the acute stage and the recovery stage were 0.61 +/- 0.12 mg/L and 0.64 +/- 0.14 mg/L, respectively, indicating no significant difference. CONCLUSION: Serum Cys-C is considered as a favorable index of GFR in children because it is not influenced by age and sex. As it is not influenced by the prerenal factors either, serum Cys-C is useful in children over 1-years-old.

A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis.

BACKGROUND: We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS: DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS: The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS: PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.

Lactoferrin in whole gut lavage fluid as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived proteins.

OBJECTIVES: We investigated which neutrophil-derived proteins in whole gut lavage fluid (WGLF) most accurately reflect disease activity in inflammatory bowel disease. METHODS: WGLF was obtained from patients undergoing whole gut lavage as a bowel preparation for colonoscopy. Twenty-seven patients with ulcerative colitis (UC), 23 patients with Crohn's disease (CD), and 35 control subjects were examined. The concentrations of lactoferrin, polymorphonuclear neutrophil elastase (PMN-E), myeloperoxidase, and lysozyme in WGLF were measured by ELISA. For the assessment of stability, WGLF samples were stored at 37 degrees C for various periods. RESULTS: In UC, the concentrations of lactoferrin, myeloperoxidase, and lysozyme in WGLF had good correlations with colonoscopic grading. Zero, 12, five, and 10 of 28 samples from active UC patients showed normal concentrations of lactoferrin, PMN-E, myeloperoxidase, and lysozyme, respectively. In CD, the concentrations of lactoferrin and myeloperoxidase had good correlations with the Crohn's disease activity index. Thirteen and seven of 36 samples from inactive CD patients (Crohn's disease activity index < or = 150) showed high concentrations of lactoferrin and myeloperoxidase, respectively. Most of them (11/13, 6/7) were found to have ulceration by colonoscopy or small bowel x-ray. The ratio of the lactoferrin concentration in the WGLF supernatant to that in total WGLF was highest among these proteins in all disease groups and control subjects. Lactoferrin and myeloperoxidase showed good stability in WGLF, whereas PMN-E and lysozyme did not. CONCLUSION: Lactoferrin is the most suitable of these proteins for use as a neutrophil-derived WGLF marker of intestinal inflammation.

Evaluation of oxidized alpha-1-antitrypsin in blood as an oxidative stress marker using anti-oxidative alpha1-AT monoclonal antibody.

BACKGROUND: alpha1-AT is a 52-kDa acute-phase protein and a typical serine proteinase inhibitor, which is present in human serum. In vivo, the inhibitor prevents tissue damage by inactivating proteinases, such as elastase, that are released from activated neutrophils in the presence of inflammation. METHODS: We obtained a monoclonal antibody against oxidized alpha1-AT(3F4) using chloramine T-oxidized alpha1-AT as the antigen. RESULTS: This antibody did not react with either the native alpha1-AT or the elastase-alpha1-AT complex. However, it reacted with alpha1-AT oxidized by various oxidants and peroxide lipid. The oxidized alpha1-AT is a polymer with a molecular mass of 100-200 kDa in addition to the 52-kDa protein that corresponds to the native alpha1-AT in sera. In vitro evaluations reveal that fatty acids are involved in the polymerization. Furthermore, the concentrations of oxidized alpha1-AT in the sera of patients with inflammatory and rheumatoid diseases were higher than those in healthy subjects. CONCLUSIONS: We considered that 3F4 is an effective antibody that can specifically recognize oxidized alpha1-AT, a marker of oxidative stress.