Modulation of Gold Nanorod Growth via the Proteolysis of Dithiol Peptides for Enzymatic Biomarker Detection.

Gold nanorods possess optical properties that are tunable and highly sensitive to variations in their aspect ratio (length/width). Therefore, the development of a sensing platform where the gold nanorod morphology (i.e., aspect ratio) is modulated in response to an analyte holds promise in achieving ultralow detection limits. Here, we use a dithiol peptide as an enzyme substrate during nanorod growth. The sensing mechanism is enabled by the substrate design, where the dithiol peptide contains an enzyme cleavage site in-between cysteine amino acids. When cleaved, the peptide dramatically impacts gold nanorod growth and the resulting optical properties. We demonstrate that the optical response can be correlated with enzyme concentration and achieve a 45 pM limit of detection. Furthermore, we extend this sensing platform to colorimetrically detect tumor-associated inhibitors in a biologically relevant medium. Overall, these results present a subnanomolar method to detect proteases that are critical biomarkers found in cancers, infectious diseases, and inflammatory disorders.

The effect of poly (ADP-ribose) polymerase inhibition on aminoglycoside-induced acute tubular necrosis in rats.

INTRODUCTION: Aminoglycosides (AG) cause nephrotoxicity in 10 - 20% of patients. One of the mechanisms is by generating reactive oxygen species (ROS), leading to DNA destruction and activation of poly(ADPribose) polymerase (PARP) causing necrotic tubular cell death. PARP inhibition on gentamicin-induced nephrotoxicity was studied. METHODS: 19 female Wistar-Kyoto rats divided into 3 groups: control (3 rats receiving no treatment); gentamicin-treated group (8 rats); and 8 rats treated with gentamicin combined with 3-aminobenzamide (3 AB). Kidney functions, protein, and gentamicin levels as well as urinary trypsin inhibitory activity (TIA) were measured. Tissue microscopic examination and immunohistochemical study for proliferative cell nuclear antigen (PCNA) were determined. The effect of PARP inhibitor on the bactericidal activity of gentamicin was also assessed. RESULTS: The following results were statistically significant: urea (mg/dL) 39.9 ± 5.86, 88.3 ± 50.3, and 48.5 ± 12.7 (p = 0.048); serum creatinine (mg/dL): 0.6 ± 0.26, 1.05 ± 0.7, 0.6 ± 0.06 (p = 0.043); proteinuria (mg/24-hours): 7.27 ± 3.65, 41.2 ± 18.1, and 17.6 ± 13.9 (p = 0.050); the number of tubular macronuclei (per 10 mm2): 18.33 ± 16.07, 218 ± 101.8, 41.7 ± 36.2 (p = 0.012); the number of dilated tubes (per 10 mm2): 61.67 ± 12.58, 276.3 ± 112.7, 140.0 ± 90.9 (p = 0.04); and the number of PCNA positive nuclei (per 10 mm2): 223.3 ± 95.69, 3,585 ± 2,215.3, 626.7 ± 236.9 (p = 0.034) in the control, gentamicin, and gentamicin+3AB-treated groups, respectively. The following biochemical and histologic parameters were also examined, however, they showed no statistically significant difference: TIA (p = 0.055), mitoses (p = 0.14), mononuclear infiltrate (p = 0.188), and intratubular cast formation (p = 0.084). No effect on bactericidal activity was observed. CONCLUSION: This study illustrates that PARP inhibitor significantly attenuates gentamicin-induced nephrotoxicity in rats with no effect on the bactericidal activity.

Effect of urinary protease inhibitor (ulinastatin) on cardiopulmonary bypass: a meta-analysis for China and Japan.

OBJECTIVES: A meta-analysis was conducted to investigate the effects of ulinastatin treatment on adult patients undergoing cardiac surgery under cardiopulmonary bypass (CPB). METHODS: Seven electronic databases were searched for reports of randomized, controlled trials conducted up to February 2014 in which patients undergoing cardiac surgery with CPB were administered ulinastatin in the perioperative period. RESULTS: Fifty-two studies with 2025 patients were retained for analysis. The results showed that the ulinastatin can attenuate the plasma levels of pro-inflammatory cytokines and enhance the anti-inflammatory cytokine levels in patients undergoing cardiac surgery with CPB. Meanwhile, the ulinastatin had a significant beneficial effect on myocardial injury. The mean differences (MD) and 95% confidence intervals (95% CI) of biochemical markers were -63.54 (-79.36, -47.72) for lactate dehydrogenase, -224.99 (-304.83, -145.14) for creatine kinase, -8.75 (-14.23, -3.28) for creatine kinase-MB, and -0.14 (-0.20, -0.09] for troponin I (all P<0.01). However, neither hemodynamics nor cardiac function improved significantly, except that the MD and 95% CI of mean arterial pressure were 2.50 (0.19, 4.80) (P = 0.03). There were no statistically significant differences in the use of inotropes, postoperative bleeding, postoperative complications, the intensive care unit (ICU) stay, and the hospital stay; however, the frequency of auto resuscitation increased significantly (OR 1.98, 95%CI 1.19 to 3.30, P<0.01), the duration of intubation (MD -1.58, 95%CI -2.84 to -0.32, P<0.01) and the duration of mechanical ventilation (MD -3.29, 95%CI -4.41 to -2.17, P<0.01) shortened significantly in patients who were treated with ulinastatin. CONCLUSIONS: Ulinastatin can reduce the plasma levels of pro-inflammatory cytokines and elevate anti-inflammatory cytokine in patients from China and Japan undergoing cardiac surgery with CPB. Ulinastatin treatment may have protective effects on myocardial injury, and can increase the frequency of auto resuscitation, shorten the duration of intubation and mechanical ventilation.

Protective effects of urinary trypsin inhibitor on vascular permeability following subarachnoid hemorrhage in a rat model.

AIMS: Inflammation and apoptosis play important roles in increasing vascular permeability following subarachnoid hemorrhage (SAH). The objective of this study was to evaluate whether urinary trypsin inhibitor (UTI), a serine protease inhibitor, attenuates vascular permeability by its antiinflammatory and antiapoptotic effects after experimental SAH. METHODS: Subarachnoid hemorrhage models were established in adult male Sprague-Dawley rats by endovascular perforation. UTI was administered by intraperitoneal injection immediately following SAH. Brain edema was assessed by magnetic resonance imaging (MRI) at 24 h after SAH. Neurological deficits, brain water content, vascular permeability, malondialdehyde (MDA) concentration, and myeloperoxidase (MPO) activity were evaluated. Immunohistochemical staining and Western blot were used to explore the underlying protective mechanism of UTI. RESULTS: Urinary trypsin inhibitor 50,000 U/kg significantly attenuated brain edema and neurological deficits and reduced vascular permeability at 24 h after SAH. MDA concentration and MPO activity in hippocampus were significantly decreased with UTI treatment. Furthermore, the levels of phosphorylated JNK, NF-κB (p65), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and proapoptotic protein p53, caspase-3 were elevated in the microvascular endothelial cells of the hippocampus after SAH, which were alleviated with UTI treatment. CONCLUSION: Urinary trypsin inhibitor reduced vascular permeability after SAH through its antiinflammatory and antiapptotic effects via blocking the activity of JNK, NF-κB, and p53.

Affinity purification of urinary trypsin inhibitor from human urine.

Affinity protocols for the purification of urinary trypsin inhibitor (UTI) were developed. To imitate the substrate/inhibitor-binding domain (S1 domain) of trypsin and chymotrypsin, the key amino acid residues were composed to sorbents. The sorbents were then subjected to adsorption analysis with UTI. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. The purified enzyme was subjected to SDS-PAGE, trypsin inhibitor activity and peptide map fingerprinting analysis. As calculated, the theoretical maximum adsorption (Q(max)) of two affinity sorbents entitled as S-D-G and S-S-G were 31.7 and 30.1 mg/g, respectively; the desorption constants K(d) of the two sorbents were 8.9 and 18.6 µg/mL, respectively. After the separation of UTI with S-D-G and S-S-G, reducing SDS-PAGE analysis revealed that the protein was a single polypeptide with the mass of ~66 kDa, and the purified proteins were ~95 and 97% pure, respectively; the band on gel was further confirmed with peptide map fingerprinting analysis. Protein and bioactivity recoveries were 1.3 and 75.9% with S-D-G, 1.0 and 70.2% with S-S-G, respectively.

Proteomic analysis of pancreatic secretory trypsin inhibitor/tumor-associated trypsin inhibitor from urine of patients with pancreatitis or prostate cancer.

The development of proteomic methods, especially mass spectrometry, has brought new possibilities to tumor marker research. Pancreatic secretory trypsin inhibitor (PSTI), a common known biomarker for various malignancies, occurs on genetic variants that we are able to detect at the protein level with proteomic techniques using immunoaffinity capture prior to liquid chromatography-mass spectrometry (LC-MS). We also show that PSTI can be detected in urine from cancer patients using a two-step peptide enrichment technique and LC-MS. These results show that tumor-associated peptides can be detected in urine by proteomic techniques.

Immunological evaluation of urinary trypsin inhibitors in blood and urine: role of N- & O-linked glycoproteins.

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-alpha-I, and I-alpha-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm-Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.

Development of an ELISA test for determination of the urinary trypsin inhibitor: analytical performance and applications.

Increased urinary excretion of urinary trypsin inhibitor (UTI) has been reported in various inflammatory conditions and in Alzheimer's subjects, but its diagnostic potential remains to be elucidated. A reliable and specific enzyme-linked immunosorbent assay (ELISA) test for the determination of the UTI in human urine was developed. This assay was performed using 96-well microtiter plates. The plate surface is coated with an anti-UTI polyclonal antibody, the urine sample was added in a dilution range, and the detection was achieved using the enzyme-conjugated antibody. The assay was quantified by the build-up of colored product upon the addition of the substrate. Recoveries were 93%, and the intra- and inter-assay CVs were 4.25% and 21%, respectively. The ELISA showed parallelism of standard and urine samples and no significant interference by the biological matrix. The usefulness of the assay has been demonstrated by applying it to urine samples from Alzheimer's disease patients, and comparing with negative controls. UTI urinary levels are significantly increased in Alzheimer's subjects.

Pathophysiology and diagnostic value of urinary trypsin inhibitors.

Inflammation is an important indicator of tissue injury. In the acute form, there is usually accumulation of fluids and plasma components in the affected tissues. Platelet activation and the appearance in blood of abnormally increased numbers of polymorphonucleocytes, lymphocytes, plasma cells and macrophages usually occur. Infectious disorders such as sepsis, meningitis, respiratory infection, urinary tract infection, viral infection, and bacterial infection usually induce an inflammatory response. Chronic inflammation is often associated with diabetes mellitus, acute myocardial infarction, coronary artery disease, kidney diseases, and certain auto-immune disorders, such as rheumatoid arthritis, organ failures and other disorders with an inflammatory component or etiology. The disorder may occur before inflammation is apparent. Markers of inflammation such as C-reactive protein (CRP) and urinary trypsin inhibitors have changed our appraisal of acute events such as myocardial infarction; the infarct may be a response to acute infection and (or) inflammation. We describe here the pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi). It is an important anti-inflammatory substance that is present in urine, blood and all organs. We also describe the anti-inflammatory agent bikunin, a selective inhibitor of serine proteases. The latter are important in modulating inflammatory events and even shutting them down.

[Comparison of the trypsin inhibitor activity in urine of donors and patients with kidney diseases]. / Sravnenie aktivnosti ingibitora tripsina v moche donorov i bol'nykh zabolevaniiami pochek.

The general and acid-stable activities, i.e. anti-tryptic activity (ATA) and anti-chemotryptic activity (ACTA), of a trypsin inhibiter in the urine (TIU) were determined for patients and donors. A lack of reliable differences between the general and acid-stable activities in donors and in patients with nephritis is indicative of that TIU is the main inhibiter of trypsin in human urine. A correlation degree between the general ATA and ACTA (r > 0.7) means that the two-domain inhibiter form is excreted into the urine. A comparison between the general and acid-stable ATAs suggests the presence of other trypsin inhibiters in the urine of patients.

Combination assay of urinary free L-fucose and trypsin inhibitor may be useful indicators of disease activity in patients with hematologic malignancies.

L-Fucose is a monosaccharide located at the non-reducing ends of sugar chains of glycoconjugates. Urinary L-fucose (U-FC) is excreted as free L-fucose, and clinically useful as a tumor marker of digestive organ cancers. We evaluated the clinical usefulness of U-FC levels in patients with various hematologic malignancies because U-FC for hematologic malignancies has only rarely been described. The mean U-FC levels in the acute nonlymphocytic leukemia (ANLL), myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD) groups were significantly higher than in the control group (P < 0.05). Recently, we reported that urinary trypsin inhibitor (UTI) levels in patients with ANLL, MDS, non-Hodgkin's lymphoma and multiple myeloma were significantly elevated, compared with those in healthy adult volunteers. Noninvasive combination assays of UTI and U-FC may have a higher accuracy in diagnosis of ANLL and MDS than those of UTI or U-FC alone. UTI and U-FC combination assays, noninvasive for patients, could be expanded as useful indicators in hematologic malignancies.

Kinetics of urinary trypsin inhibitor in patients undergoing partial hepatectomy.

BACKGROUND: The kinetics and role of urinary trypsin inhibitor (UTI) in liver surgery are unclear. We investigated the effects of preoperative liver function and the extent of liver resection on postoperative UTI synthesis in the liver after partial hepatectomy. METHODS: Sixty-one consecutive patients who underwent liver resection were the subjects of the study. Plasma and urine UTI, plasma C reactive protein (CRP) and plasma and urine creatinine were measured perioperatively. RESULTS: Although the average plasma UTI level did not change significantly, the average urine UTI level per day showed a change similar to that of the average plasma CRP level, reaching a maximum of approximately eight times the preoperative level on the second postoperative day (86,610 +/- 53,109 U/day). The maximum postoperative increase in urine UTI excretion per day (delta-uUTImax) correlated significantly with the maximum increase in CRP and the increase in creatinine clearance. Multiple regression analysis revealed that delta-uUTImax was significantly and positively correlated with the indocyanine green plasma disappearance rate and operation duration, and negatively correlated with the resection rate. CONCLUSIONS: The postoperative urine UTI level may reflect preoperative liver function and the extent of liver resection after partial hepatectomy.

Urinary trypsin inhibitor levels in the urine of patients with haematological malignancies.

The urinary trypsin inhibitor (UTI) levels in the urine of patients with various haematological malignancies were determined, using automated latex agglutination immunoturbidimetry. The mean UTI levels in urine in acute non-lymphocytic leukaemia, myelodysplastic syndrome, non-Hodgkin's lymphoma, and multiple myeloma groups were significantly elevated, compared with the normal control group. It was found that the UTI level in urine changed from an elevated value to a normal value with haematological improvement by chemotherapy in a patient with myelodysplastic syndrome included in a previous study. These results suggest tha

[Urinary trypsin inhibitor in pregnant women with normal and pathological course of pregnancy]. / Inhibitor trypsyny w moczu kobiet w ciazy fizjologicznej i patologicznej.

Urinary trypin inhibitor concentration (UTI) was measured by Fritz and all method in the groups of women: non-pregnant (I), pregnant (II), in pregnancy complicated by EPH-gestosis (III) and in the prolongated pregnancy (IV). Furthermore, in the urine from the investigated group the index protein/creatinine was established. There was noticed the statistically significant increase in the UTI concentration in II, III, IV groups comparing to the non-pregnant group--I, and the increase in UTI in III and IV group in comparison to group II. The UTI measurement in the pregnant women connected with the index protein/creatinine could be significant for diagnosing of the pathology of pregnancy.

Urinary excretion of Bowman-Birk inhibitor in humans after soy consumption as determined by a monoclonal antibody-based immunoassay.

The Bowman-Birk inhibitor (BBI) found in soybeans is a serine protease inhibitor with anticarcinogenic activity. In the present study, an ELISA for BBI was developed with the use of a monoclonal antibody against a reduced form of BBI. This newly developed ELISA method was used to measure the urinary levels of BBI metabolites in nine human subjects after consumption of 36-oz or 60-oz soymilk (containing 105 or 175 mg of BBI) at two time points 36 h apart. The results demonstrate that urinary BBI excretion rates peaked within 6 h and decreased to baseline levels within 12-24 h after soymilk ingestion. The changes in BBI:creatinine ratios in urine closely paralleled the changes in urinary BBI excretion rates after soymilk consumption. These data suggest that BBI ingested p.o. is absorbed and could be bioavailable for cancer chemoprevention in other organs in addition to those in the gastrointestinal tract.

[Supplement of ulinastatin on renal function after cardiopulmonary bypass].

The effects of Miraclid (ulinastatin) on renal tubular function after open thorax surgery under cardiopulmonary bypass were investigated. On the 3rd day after open thorax surgery, which had lasted more than 127 min under cardiopulmonary bypass, the levels of urinary ulinastatin in the Miraclid group and control (without Miraclid) were 170 IU.mg Cr-1 and 95 IU.mg Cr-1, respectively. In the Miraclid group, 300,000 units.day-1 of Miraclid was administrated for three postoperative days. N-acetyl-beta-d-glucosaminidase in urine as a marker of tubular function rose significantly on the seventh postoperative day in the control group but not in patients with Miraclid group. These data suggested that Miraclid 300,000 units.day-1 was needed to protect renal tubular function and more than that dose was needed to prevent the deterioration of renal function after open thorax surgery after cardiopulmonary bypass lasting more than 127 min.

Single-dose administration of Bowman-Birk inhibitor concentrate in patients with oral leukoplakia.

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor and a potential cancer chemopreventive agent for humans. In this Phase I clinical trial, BBI concentrate was administered as a single oral dose to 24 subjects with oral leukoplakia. Pharmacokinetics of BBI was analyzed, and subjects were monitored clinically for toxic effects. Subjects received between 25 and 800 chymotrypsin inhibitor units (CIU) of the compound in a dose escalation trial. BBI was taken up rapidly, and a metabolic product of BBI was excreted in the urine within 24-48 h. No clinical or laboratory evidence of toxicity was observed in the study. Protease activity was also measured in buccal cells to evaluate usefulness as a biomarker. Single-dose BBI concentrate administered up to 800 CIU was well tolerated and appeared to be nontoxic. Further investigation in Phase II clinical trials is being done.

Detectable concentrations of urinary trypsin inhibitor in cerebrospinal fluid.

Urinary trypsin inhibitor (UTI) is a physiological protease inhibitor produced in the liver and excreted into urine. Urinary trypsin inhibitor-like antigen has been demonstrated on glial cells in the brain. This study measured cerebrospinal UTI levels in various conditions. Seven subjects in each of the following groups were studied: patients undergoing spinal anesthesia for minor surgery, spinal anesthesia for cesarean section, removal of meningioma, or at postoperative day 3 after ruptured intracranial aneurysm clipping. Cerebrospinal fluid was collected from a spinal needle, a needle puncturing the sylvian fissure, or a drainage tube from the optical carotid cistern. Urine was collected from a urinary catheter. Cerebrospinal and urinary UTI concentrations were measured by radioimmunoassay, and the urinary UTI concentration was divided by urinary creatinine concentration to give the systemic UTI concentration. The cerebrospinal UTI concentration in the brain tumor and postoperative state groups was significantly higher than in the spinal anesthesia and cesarean section groups. The systemic UTI concentration in the cesarean section and postoperative state groups was significantly higher than in the spinal anesthesia and brain tumor groups. The present results demonstrate that UTI can be detected in the cerebrospinal fluid, and suggest that cerebrospinal UTI increases in patients with a brain tumor or inflammation and is not affected by systemic UTI.