Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0.5 % smaller. In addition, we investigated the pressure dependence of the apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.     

CA 125 in biological fluids

CA 125 is not a specific tumor marker, and is synthesized by normal and malignant cells of different origin (mainly in tissues derived from the müllerian epithelia) in a similar proportion. Abnormal CA 125 levels may be found in fluids of different origin (ascites, pleura, pericardium, amniotic fluid, cyst fluid, bronchoalveolar fluid, etc.) and in serum from patients with these fluids. Differences in serum CA 125 found in malignant or benign diseases may be related to the number of cells that synthesize the marker, and are highly dependent on the access to serum, where the marker is normally determined. Moreover, CA 125 is a very good tumor marker in ovarian and lung cancer. The sensitivity of CA 125 in ovarian cancer is related to stage (40-95%), histological type (lower levels in mucinous adenocarcinoma), and the marker is useful in the early detection of recurrence (sensitivity 80%) and in therapy monitoring. It's sensitivity in lung cancer is lower than in ovarian cancer, 39% in locoregional malignancies and 69% in metastatic disease, but clearly related to stage and histology (mainly in adenocarcinomas and large cell lung cancer) and it is useful in prognosis and disease monitoring.     

Biomarker discovery in biological fluids

Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development.     

The NMR study of human biological fluids for detection of pathologies NMR study of biological fluids

The paper deals with the NMR spectra obtained using preparations of five different human biological body fluids. Characteristic metabolite signals of blood, urine, tears, saliva, and sweat spectra have been determined and classified. The biological body fluid samples were used for search and identification of biomarkers of cardiovascular disease. Absolute functional biomarkers for diseases such as coronary heart disease (CHD) have not been recognized even in the case acute myocardial infarction. A hypothesis explaining reasons of lack of such markers has been formulated. The results of comparative analysis of blood and urine samples from humans and some laboratory animals are given. Identify and analyze signals of metabolites of pathogenic microflora and their dynamics in the urine from patients with urogenital diseases have been determined and analyzed and characteristic biomarkers have been recognized.     

Measurement of peptidoleukotrienes in biological fluids

Samples of human bronchoalveolar lavage fluid (BALF) and urine were utilized to demonstrate methods for quantitation and validation of leukotrienes (LTs). These methods utilize an enzyme immunoassay (EIA) that uses commercially available reagents, the antibody recognizing LTC4, LTD4, LTE4, and N-acetyl LTE4. BALF containing epithelial lining fluid was collected from atopic asthmatics both before and 5 min after the subjects had been challenged with a local instillation of allergen into the airways. BALF samples collected without allergen challenge had low levels of immunoreactive LTs, whereas samples collected after allergen were markedly elevated. After high-performance liquid chromatography (HPLC) separation of LTs, EIA revealed the presence of LTC4. The identity was validated by incubating LTC4 with a bovine gamma-glutamyl transpeptidase with dipeptidase activity that converted added [3H]-LTC4 as well as LTC4 immunoreactivity to LTE4. Urine samples collected from six healthy volunteers, one patient with adult respiratory distress syndrome (ARDS), and three patients in status asthmaticus were also analyzed for LTs. After HPLC separation of LTs and quantitation by EIA, urine samples from healthy subjects were found to have low but measurable LTE4. In contrast, the urine samples from the patients in status asthmaticus and from the ARDS patient had large elevations of LTE4 levels compared with healthy subjects. When the HPLC fractions containing [3H]LTE4 and LT immunoreactivity in the ARDS sample were treated with acetic anhydride, HPLC analysis indicated that both radiolabel and immunoreactivity now eluted at the retention time of N-acetyl LTE4, the derivatized product of LTE4. The methods described are relatively easy and can be used to measure and validate the existence of peptidoleukotrienes in biological samples.     

Measurement of dimethylglycine in biological fluids

The method of quantitating N,N-dimethylglycine involves cation-exchange high-performance liquid chromatography and detection of dimethylglycine with dimethylglycine dehydrogenase. Dimethylglycine was added to plasma and urine and samples were assayed for dimethylglycine. Plasma and urine to which no dimethylglycine was added were also assayed. Recoveries of added dimethylglycine were 99 to 104% with no endogenous dimethylglycine found in rat plasma or normal human urine. The human plasma used contained a small amount of endogenous dimethylglycine. The cation-exchange chromatography separates dimethylglycine from other compounds which can serve as substrates for dimethylglycine dehydrogenase. Repeatability of the assay is +/- 10%. Using this method we have identified dimethylglycine in the urine of a 1-month-old female human patient.     

Ensembles of synthetic polymers mimic biological fluids

Recently a report by Ruan et al. in Nature described how relatively simple random heteropolymers can replicate the properties of biological fluids. These polymers capture the segmental-level interactions between proteins and could enhance folding of membrane proteins, improve stability, and enable DNA sequestration in a chemistry specific manner.     

Measurement of lonized magnesium in biological fluids

A method that provides for direct in vitro measurement at 37°C of ionized magnesium in biological fluids using the Abbot bichromatic analyzer, a fully automated instrument is described. The metallochromic dye Eriochrome blue SE was used to measure ionized magnesium levels in Mueller-Hinton agar fluids. Formulation of an appropriate blank was found necessary and was made by treating agar fluids with Chelex 100 to remove divalent cations. The influence of pH in the biological range was examined and poses a significant but manageable source of error. The method allowed an appropriate assessment of the role of ionized magnesium in the susceptibility testing of Pseudomonas aeruginosa on Mueller-Hinton agar with the aminoglycoside antibiotics.