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PubMed1995

Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries.

A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

Animals↗
PubMed2001

Quantitative analysis of herpesvirus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR.

Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogeneous (range 2-20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001-170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5-4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

Amino Acid Sequence↗
PubMed1996

Quantitative analysis of the spinal cord motoneuron under chronic compression: an experimental observation in the mouse.

We investigated quantitative changes in spinal cord motoneurons following chronic compression using a mouse model of cervical cord compression. Twenty-five tip-toe-walking Yoshimura (twy) mice with calcified mass lesions compressing the spinal cord posterolaterally at the C1-C2 vertebral levels were compared with five Institute of Cancer Research (ICR) mice that served as controls. Spinal cord motoneurons in the anterior grey horn between the C1 and C3 spinal cord segments were Nissl-stained and counted topographically and then analysed in relation to the extent of spinal cord compression. The number of motoneurons in C1-C3 spinal cord segments decreased significantly with a linear correlation with the transverse area of the spinal cord when the cord was compressed to 50-70% of control values. A significant reduction in the number of motoneurons occurred at the C2-C3 spinal cord segment compressed at the C1-C2 vertebral level. In contrast, at the level rostral to the C1 vertebra, the number of motoneurons increased significantly in proportion to the magnitude of compression. The current study demonstrates that a number of neurons, morphologically consistent with anterior horn cells, were observed at a rostral site absolutely free of external compression where no such cells normally exist.

Animals↗
PubMed1989

Quantitative analysis of ventral denticular patterns of Drosophila melanogaster larvae and the regulation of the bithorax complex.

A quantitative model of the effect of the bithorax complex on segmentation is presented which could explain the known data of the spatiotemporal regulation of key gene complex during early Drosophila development, in relation to their effects on some of the segmentation landmarks. The model tries to put together the two different genetic levels, the genotypic and the phenotypic. At the genotypic level, a minimal cross-regulatory network of the different genes involved, Antp, Ubx, abd-A and Abd-B which explains the reported levels of expressions of these genes. At the phenotypic level, the pattern of the ventral denticle belts across the larva which are characteristics of the different segments have been compared by calculating a value of the degree of similarity in the case of the wild-type and several mutant combinations. Finally the two parts of the model are combined, showing that a satisfactory agreement between the two can be achieved. Therefore, this work is a first attempt to develop a method which will provide an explanatory solution of the old question in morphogenesis of how the phenotype is directed by the genotype of a cell or organism.

Animals↗
PubMed2002

Quantitative analysis of technical polymer mixtures by matrix assisted laser desorption/ionization time of flight mass spectrometry.

We report quantitative MALDI-TOF measurements for polydimethylsiloxane (PDMS) of two different molecular weights using the relative ratio of the signal intensities of integrated oligomer distributions for these two molecular weight distributions. By reporting the ratio of intensities of the integrals of two oligomer distributions, we assume that the ionization and desorption efficiencies, crystallization conditions and other factors affecting intensity are similar. Poly(methyl methacrylate) (PMMA-33,000) was mixed with PDMS samples to show whether the presence of another material might affect the desorption efficiency. Quantitative values for the number-average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersities (D) were calculated using the oligomer distributions. The results show a linear relationship between the analyte concentrations and the signal intensities in the range from 1,000 Da to 10,000 Da, and the desorption efficiency of these two PDMS materials was the same even in the presence of PMMA.

Journal Article↗
PubMed2004

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.

Cell Line↗
PubMed1998

Quantitative analysis of C cells in Hashimoto's thyroiditis.

The objective of the present investigation was to study quantitatively C cells in Hashimoto's thyroiditis (HT) by analyzing 22 thyroids obtained at autopsy or thyroidectomy from 16 patients with thyroiditis and from 6 normal subjects. Six different areas were sampled on average and labeled with a monoclonal anticalcitonin antibody by immunohistochemistry using the avidin-biotin-peroxidase complex. Normal thyroids from adult patients with no thyroid disease were used as control. C cells were counted in 1 cm2 fields and the mean number of cells per field was calculated. Data were analyzed statistically by the Mann-Whitney test. The inflammatory process detected in cases of HT was usually moderate (50%) and the number of C cells ranged from 0 to 12.2 per 1 cm2 field. The number of C cells in normal thyroids ranged from 20 to 148 per 1 cm2 field, with a median of 35.2 cells per field. The results demonstrate a significant decrease in C cell number in HT compared with normal thyroids, indicating that the inflammatory process causes destruction of both follicular and C cells, which are replaced by fibrosis.

Calcitonin↗
PubMed1989

Cutaneous blood flow and percutaneous absorption: a quantitative analysis using a laser Doppler velocimeter and a blood flow meter.

Cutaneous blood flow has been directly quantitated in vivo for the first time without animal death utilizing the rat skin sandwich flap. This was accomplished by conducting experiments that made a direct correlation between two instruments: a laser Doppler velocimeter and an electromagnetic blood flow meter. Data demonstrate that the correlation between these two instruments is high and reproducible (r = 0.96) with a small (1.3%) coefficient of variation. Blood flow to skin in the unmanipulated state varies from 0.7 to 1.2 mls/min in an anesthetized rat. Application of the blood flow correlation to the determination of percutaneous absorption of caffeine across human skin and benzoic acid across rat skin demonstrates that assuming cutaneous blood flow is a particular value day to day in any skin type results in an apparent wide range of total compound absorbed across that skin on independent occasions. Utilizing actual blood flow measurements to calculate the amount of chemical absorbed reduces the range of variability in the total amount of chemical absorbed and provides a more accurate knowledge of events occurring during a particular time of the absorption process. Quantitation of cutaneous blood flow will be useful in physiologic and pharmacologic studies where actual cutaneous blood flow is likely to be important to the processes studied, e.g., delivery of drug to skin, metabolism within the skin, and disposition of drug to blood and skin following topical drug application.

Animals↗
PubMed2002

Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions.

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.

Bacillus cereus↗
PubMed1995

Intracranial arteriovenous malformations: quantitative analysis of magnitude contrast MR angiography versus gradient-echo MR imaging versus conventional angiography.

PURPOSE: To quantitatively analyze magnitude contrast (MC) magnetic resonance (MR) angiography and gradient-echo (GRE) MR imaging for evaluation of persistent transnidal blood flow in intracranial arteriovenous malformations (AVMs) of various sizes before or after stereotaxic radiosurgery. MATERIALS AND METHODS: Thirty-three patients with AVMs underwent 42 MC MR angiographic (maximum intensity projection [MIP] and source imaging), GRE imaging with and without flow compensation, and conventional angiographic studies within 90 days of each other. RESULTS: For MIP, source, and GRE images, the sensitivities for detection of large AVMs (> 1 cm) were 95%, 100%, and 100%, respectively, and for small AVMs (< or = 1 cm) were 27%, 50%, and 67%, respectively. MC MR angiography and GRE imaging depicted all eight nidi that were completely thrombosed on conventional angiograms. CONCLUSION: GRE imaging and MC MR angiography reliably depict AVMs larger than 1 cm. GRE is more sensitive for detection of small residual nidi (< 1 cm). Source images must be evaluated to accurately assess low-flow lesions. Neither GRE nor MR angiography is reliable for detection of mean nidus diameters less than 0.36 cm.

Cerebral Angiography↗
PubMed1994

Quantitative analysis of changes of automated perimetric thresholds after pupillary dilation and induced myopia in normal subjects.

We quantitatively analyzed the changes of the (retinal) threshold sensitivities between normal status and dilated emmetropic status, and between dilated emmetropic status and -1.0D induced myopic status. Three consecutive visual field examinations by Humphrey Field Analyzer C-30-2 threshold test and STATPAC program were performed in 36 eyes of 21 normal subjects who ranged in age from 23 to 40 years. The results were as follows: 1) There was a statistically significant decrease (0.87 +/- 0.91dB)in the mean threshold sensitivity within the central 30 degrees at the dilated emmetropic status (27.90dB) as compared with the normal status (28.77dB)(p = 0.0001). 2) There was a statistically significant decrease(0.99 +/- 0.61dB) at -1.0D induced myopic status(26.91dB) as compared with the dilated emmetropic status(27.90dB)(p = 0.0001). 3) After dilation, the mean threshold sensitivity between 20 and 30 degrees of field showed statistically significant decrease as compared with those within the central 20 degrees of visual field(p < 0.05). In case of -1.0D induced myopia, there were uniform decreases in the mean threshold sensitivities within the central 30 degrees of visual field (p > 0.05). Therefore, we emphasize the importance of consistent pupillary size and correct refractive state in performing the serial automated visual field tests.

Adult↗
PubMed1975

Quantitative analysis of tryptophan metabolites in urine of normal and endotoxin poisoned mice.

We have developed methods for quantitatively monitoring the in vivo flow of tryptophan between the kynurenine and serotonin pathways. Our data suggest that endotoxin-poisoned mice have more tryptophan flowing into the serotonin pathway than normal mice both with and without tryptophan load. It appears that the distribution of tryptophan between the serotonin and kynurenine pathways in vivo is constant over a very wide dose range and that substrate availability rather than tryptophan oxygenase activity determines the precise amounts of tryptophan which flows into the two pathways.

Animals↗
PubMedDate not supplied

The effective mass concept: theoretical and quantitative analysis of the forbidden indicator phenomenon.

This paper describes the concept of effective mass, which provides a quantitative explanation for the forbidden indicator phenomenon. The forbidden indicator phenomenon pertains to the problems encountered when the concentration of an indicator used to measure volume or flow by the indicator-dilution technique approaches the concentration of the solution to which it is to be added. This phenomenon is of particular concern when applying the saline-dilution method with hypertonic saline, to measurement of cardiac output. Clinically, one would like to give as little additional salt to a patient as possible while maintaining a signal-to-noise ratio adequate to give reliable results. This paper develops an equation that permits accurate calculation of volume or flow by the indicator-dilution method, even when indicator solutions with concentrations approaching that of the original solution are used (i.e., the forbidden indicator). The validity of this equation is demonstrated experimentally using the saline-dilution method to measure flow in a hydraulic model of the circulation.

Biomedical Engineering↗
PubMed1975

An improved method for the quantitative analysis of M-mode echocardiograms.

A computer-assisted system is described which speeds and extends the quantitative interpretation of M-mode echocardiographic recordings. The system consists of a digitizing tablet, minicomputer, TV monitor and a hard copy device. M-mode echocardiograms are placed on the digitizing surface and traced using the digitizing pen. The entered signal includes the endocardial surfaces of the anterior and posterior left ventricular wall for at least one cycle, and two Q waves from a simultaneously recorded ECG to identify end diastole and heart rate. End systole is determined automatically as corresponding to the minimum LV dimension. Results of analysis include continuous plots of estimated volume and circumferential fiber shortening rate (CFSR) vs time. Determinations of special interest are also displayed: enddiastolic volume (EDV) and endsystolic volume (ESV), ejection fraction, cardiac output, mean and peak CFSR. M-mode echocardiograms obtained from 25 normal volunteers are used to evaluate the system. The standard error of the estimate of the computer-assisted system is comparable to the error between observers, furthermore the computer system adds no significant systematic or random error. Comparison between M-mode estimated volumes and angiographically determined values has been described previously and Sy - x here is significantly greater. The main advantages of this system are: 1. a continuous plot of estimated LV volume and CFSR is provided; 2. beat-to-beat analyses are facilitated; 3. the automatic determination of end systole removes possible errors in judgement made previously; 4. it is time saving when one considers the amount of data obtained. With these advantages and the generally satisfactory performance in the clinical trials, this system appears to have extended the clinical quantitative capabilities of M-mode echocardiograms.

Adolescent↗
PubMed2003

Quantitative analysis of kallikrein 15 gene expression in prostate tissue.

PURPOSE: The newly discovered human kallikrein 15 gene KLK15 has been shown in preliminary analysis to be associated with more aggressive types of prostate cancer. We quantitatively measured and compared gene expression of KLK15 in malignant and benign prostate tissues. MATERIALS AND METHODS: Matched prostate tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 90 patients who underwent radical prostatectomy. Quantitative reverse transcriptase-polymerase chain reaction using SYBR Green I and the LightCycler system (Roche Applied Science, Mannheim, Germany) was performed. Associations of KLK15 expression with clinicopathological parameters were analyzed. RESULTS: KLK15 over expression in cancerous versus noncancerous tissue was found in 76 of the 90 patient samples (84.4%, p <0.001). The ratio of cancerous-to-noncancerous KLK15 expression tended to be higher in patients with stage pT3/4 versus pT2 tumors (p = 0.1). KLK15 expression tended to be higher in grade 3 than in grade 2 tumors and in Gleason score 7 or greater than in Gleason score less than 7 tumors (p = 0.18 and 0.23, respectively). A 1.7 cutoff at the 40th percentile provided a significant difference in stages pT2 and pT3/4 tumors (p = 0.029). CONCLUSIONS: On quantitative real-time polymerase chain reaction KLK15 expression was significantly higher in cancerous than in noncancerous tissue. Up-regulation of the KLK15 gene in advanced and more aggressive tumors may indicate a possible role for KLK15 protein as future serum marker for prostate cancer and for distinguishing tumor aggressiveness.

Actins↗
PubMed1997

A qualitative and quantitative analysis of the entorhinal cortex in schizophrenia.

The entorhinal cortex (ERC) has been implicated in schizophrenia by a number of studies. There is anatomical observation of neuronal heterotopias in the rostral ERC, which is consistent with a hypothesis of neurodevelopmental abnormalities in this disease. In view of the significant cytoarchitectonic variation of the ERC throughout its rostro-caudal extent, we performed a detailed subareal analysis of the rostral two-thirds of the entorhinal cortex (ERCr) in 14 postmortem schizophrenic brains and 14 matched controls (mean ages of 48 and 47 respectively). This systematic evaluation included both a qualitative microscopic analysis of morphogenetic anomalies that would be consistent with neurodevelopmental pathology and quantitative measurements of total neuronal number, average neuronal density, laminar volume and laminar depth from the cortical surface in cytoarchitectonically matched subareas of schizophrenic and control brains. Parcellation of the entire ERC on the basis of cytoarchitectonic criteria identified five distinct regions, similar to those described in the macaque, except that in the human brain three of the regions were further divisible into two or three subareas, yielding nine distinct cellular compartments. Five rostral areas, prorhinal (Pr), lateral (28L), intermediate rostral and caudal (281r and 281c), and sulcal (28S), comprise the ERCr. Gross and microscopic examination of these subdivisions throughout the ERCr failed to reveal laminar disorganization in any of the schizophrenic brains. The brains also did not differ significantly with respect to total neuronal number, total volume and neuronal density per laminar and subareal subdivision, or laminar thickness per entorhinal subarea. However, neuronal number and density were reduced by 12-18% in Pr and 28L, suggesting that mild quantitative abnormalities may exist in the ERCr and might possibly be revealed in a larger sample of schizophrenic brains. We have failed to confirm previous reports of laminar disorganization in the ERCr in brains of patients with schizophrenia; to the extent that this region is implicated in schizophrenia, the structural changes are likely to consist of more subtle cellular disturbances.

Adult↗
PubMed1996

Quantitative analysis of periodontal defects in a skull model by subtraction radiography using a digital imaging device.

This paper describes a quantitative study of periodontal defects produced in a skull model using subtraction images obtained with a digital imaging device, radiovisiography (RVG). Analysis using radiovisiography was compared to changes in weight and volume determined by physical measurements. Four types of periodontal defects were studied: 2-wall, 3-wall, crater, and furcation. All defects were fabricated on posterior teeth in alveolar bone and done with triplicate samples. Defects were made progressively larger in approximately 1 mm steps allowing examination of defects ranging from 1 mm to 5 mm. Radiographic images before and after each step were obtained with a radiovisiography system and subtracted. Changes in weight and defect volume were also recorded and compared to the results obtained after each step from the digital subtraction. For each defect type, a regression analysis was performed to compare changes in bone mass determined by weight with calculated bone loss determined by subtraction radiography or by volume measurements. When the calculated bone losses were compared to the true bone losses it was evident that the subtraction method frequently, but not always, underestimated the lesion sizes. For all lesions the average underestimation was 22%. The largest underestimation occurred with furcation lesions where the measured bone loss was underestimated on average by 67%. Two-walled lesions were underestimated by 30%, 3-wall lesions by 3%, and crater lesions were overestimated by 10%. Furthermore, the accuracy of each 1 mm step in bone loss varied considerably. At present, the imaging system is not sufficiently accurate to establish absolute determinations of the bone loss, but would be clinically useful in determining relative changes in bone loss or gain after treatment. In addition, caution must be taken in interpreting a given change in calculated bone loss, since considerable variation may result in either underestimation or overestimation of bone loss.

Alveolar Bone Loss↗
PubMed1994

Quantitative analysis of steroid hormone receptors and their messenger ribonucleic acids.

Ligand binding will remain the basic technique for receptor determinations in many laboratories. It can easily be set up and it is inexpensive. It has, however, the disadvantage of requiring relatively large amounts of tissue or cultured cells, if the proper multipoint measurements for Scatchard plots are to be done. Furthermore, each of the bound/free separation techniques mentioned above has its own caveat that has to be respected if analytically correct results are to be obtained. The great advantage of receptor immunoassays is their technical simplicity and the possibility of measuring a single dose of a receptor sample (in contrast to the Scatchard plot approach in ligand-binding assays). The possibility of using a single dose (even if assayed in duplicate) is a very valuable feature in all instances when only small tissue samples are available for assay. It would be very attractive for many laboratories to set up their own receptor immunoassays. The availability of suitable antibodies may not be a major obstacle. However, the necessity of possessing a supply of a highly purified receptor standard preparation may pose a problem. This is why commercial kits seem to be used so frequently. The analysis of receptor mRNA is a complement of or alternative to receptor quantitation. It must be realized, however, that special skills, as well as a molecular biology laboratory environment and equipment, are required for successful analytic work in this area. Solution hybridization is to be preferred as an approach to obtain results of a quantitative character. However, the specificity of hybridization should be checked by Northern blots. The same is true for dot/slot hybridization, which is a suitable method for semiquantitative assessments of a series of samples. Last but not least, the in situ hybridization provides invaluable information on the tissue and cell distribution of the mRNA analyzed.

Animals↗