Comparative assessment of continuum-scale models of bimolecular reactive transport in porous media under pre-asymptotic conditions.

We compare the ability of various continuum-scale models to reproduce the key features of a transport setting associated with a bimolecular reaction taking place in the fluid phase and numerically simulated at the pore-scale level in a disordered porous medium. We start by considering a continuum-scale formulation which results from formal upscaling of this reactive transport process by means of volume averaging. The resulting (upscaled) continuum-scale system of equations includes nonlocal integro-differential terms and the effective parameters embedded in the model are quantified directly through computed pore-scale fluid velocity and pore space geometry attributes. The results obtained through this predictive model formulation are then compared against those provided by available effective continuum models which require calibration through parameter estimation. Our analysis considers two models recently proposed in the literature which are designed to embed incomplete mixing arising from the presence of fast reactions under advection-dominated transport conditions. We show that best estimates of the parameters of these two models heavily depend on the type of data employed for model calibration. Our upscaled nonlocal formulation enables us to reproduce most of the critical features observed through pore-scale simulation without any model calibration. As such, our results clearly show that embedding into a continuum-scale model the information content associated with pore-scale geometrical features and fluid velocity yields improved interpretation of typically available continuum-scale transport observations.

Characterization and genetic study of the ovine alphaS2-casein (CSN1S2) allele B.

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine alpha ( s2 )-casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp(75) --> Tyr(75) and Ile(105) --> Val(105). The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp(75) --> Tyr(75) substitution modifies the protein electric charge, milk properties may result affected to some extent.

Short communication: Carora cattle show high variability in alpha(s1)-casein.

The objective of this study was to analyze the genetic variability of milk proteins of the Carora, a shorthorned Bos taurus cattle breed in Venezuela and in other Southern American countries that is primarily used for milk production. A total of 184 individual milk samples were collected from Carora cattle in 5 herds in Venezuela. The milk protein genes alpha(s1)-casein (CN) (CSN1S1), beta-CN (CSN2), kappa-CN (CSN3), and beta-lactoglobulin (LGB) were typed at the protein level by isoelectrofocusing. It was necessary to further analyze CSN1S1 at the DNA level by a PCR-based method to distinguish CSN1S1*G from B. Increased variation was found in particular at the CSN1S1 gene, where 4 variants were identified. The predominant variant was CSN1S1*B (frequency = 0.8). The second most common CSN1S1 variant was CSN1S1*G (0.101), followed by CSN1S1*C (0.082). Moreover, a new isoelectrofocusing pattern was identified, which may result from a novel CSN1S1 variant, named CSN1S1*I, migrating at an intermediate position between CSN1S1*B and CSN1S1*C. Six cows carried the variant at the heterozygous condition. For the other loci, predominance of CSN2*A2 (0.764), CSN3*B (0.609), and LGB*B (0.592) was observed. Haplotype frequencies (AF) at the CSN1S1-CSN2-CSN3 complex were also estimated by taking association into account. Only 7 haplotypes showed AF values >0.05, accounting for a cumulative frequency of 0.944. The predominant haplotype was B-A2-B (frequency = 0.418), followed by B-A2-A (0.213). The occurrence of the G variant is at a rather high frequency, which is of interest for selection within the Carora breed because of the negative association of this variant with the synthesis of the specific protein. From a cheese-making point of view, this variant is associated with improved milk-clotting parameters but is negatively associated with cheese ripening. Thus, milk protein typing should be routinely carried out in the breed, with particular emphasis on using a DNA test to detect the CSN1S*G variant. The CSN1S*G allele is likely to have descended from the Brown Swiss, which contributed to the Carora breed and also carries this allele.

Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms.

The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of alpha(S1)-casein, beta-casein, kappa-casein, and beta-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the kappa-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr(136) and Ala(148). This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.

Casein haplotype structure in five Italian goat breeds.

The aim of this work was to investigate the genetic structure of the casein gene cluster in 5 Italian goat breeds and to evaluate the haplotype variability within and among populations. A total of 430 goats from Vallesana, Roccaverano, Jonica, Garganica, and Maltese breeds were genotyped at alphas1-casein (CSN1S1), alphas2-casein, (CSN1S2), beta-casein (CSN2), and kappa-casein (CSN3) loci using several genomic techniques and milk protein analysis. Casein haplotype frequencies were estimated for each breed. Principal component analysis was carried out to highlight the relationship among breeds. Allele and haplotype distributions indicated considerable differences among breeds. The haplotype CSN1S1*F- CSN1S2*F-CSN3*D occurred in all breeds with frequencies >0.100 and was the most common haplotype in the Southern breeds. A high frequency of CSN1S1*0-CSN1S2*C-CSN3*A haplotype was found in Vallesana population (0.162). Principal component analysis clearly separated the Northern and Southern breeds by the first component. The variability of the caprine casein loci and variety of resulting haplotypes should be exploited in the future using specific breeding programs aiming to preserve biodiversity and to select goat genetic lines for specific protein production.

Single nucleotide polymorphisms in the ovine casein genes detected by polymerase chain reaction-single strand conformation polymorphism.

Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the simultaneous typing of several alleles at casein loci, as well as the detection of unknown polymorphisms. Here we describe the usefulness of the PCR-SSCP technique for casein typing in sheep. In particular, three single-nucleotide polymorphisms (SNP) are described at CSN1S1, CSN2, and CSN3, all resulting in amino acid exchanges. At CSN1S1, a transition T-->C was found, resulting in the deduced amino acid exchange Ile186-->Thr186. A transition A-->G resulting in the deduced amino acid exchange Met183-->Val183 was identified at CSN2. The 2 SNP showed a rather high frequency (ranging from 0.12 to 0.26) in 3 Italian breeds (Sarda, Comisana, Sopravissana). Another transition C-->T (Ser104-->Leu104) was found at CSN3 in one heterozygous animal.

Polymorphism of bovine MHC class I genes. Joint report of the Fifth International Bovine Lymphocyte Antigen (BoLA) Workshop, Interlaken, Switzerland, 1 August 1992.

The objectives of the Fifth International BoLA Workshop were to: standardize nomenclature, compare typing methods, and characterize BoLA haplotypes. The workshop was based on the distribution of blood samples (cells) from 60 selected cattle to 14 laboratories. Results for the class I (BoLA-A) region are presented in this paper while results for the class II regions are presented in a separate report. Thirty-six of the 50 previously established serological class I specificities were represented in the cell panel. However, only 30 specificities could be confirmed. Two specificities, A16 and A32, were upgraded from provisional, workshop (w) specificities to BoLA-A locus specificities and three new specificities, w51(w28), w52 and w53(w28), were defined. The 39 specificities distinguished 30 class I haplotypes in the 60 animals. Class I isoelectric focusing proved to be a useful adjunct to the serology. Isoelectric focusing confirmed several serologically defined splits and detected splits of A15(A8), A18(A6) and A22(w49) that had not been detected by serology. Subsequently, serological support for splits of A15(A8) and A22(w49) was found.

Measurement of alkaline phosphatase activity in serum with N-methyl-D-glucamine as a buffer: evaluation of the method for routine use.

Some aspects of the measurement of alkaline phosphatase activity concentration in human serum, using N-methyl-D-glucamine as a buffer, were evaluated with a view to the possible routine use of the method. The evaluated characteristics included: the temperature-dependence of the pH of the buffer; the effect of adding the magnesium/zinc ions buffer; the effect of the serum volume fraction; the substrate-starter versus the serum-starter mode; the effect of modifying the formulation of reagents; the within-run and the between laboratory imprecision; the correlation with an alternative routine method. Also, sex- and age-related reference values were produced, based on 2968 values from selected reference sample groups in 7 laboratories. In general, the results demonstrate the robustness of the method, its adaptability to a variety of mechanized analysers, and hence its feasibility as a routine measurement method.

In vitro and in vivo behaviour of narciclasine released from matrices based on poly (2-hydroxyethyl methacrylate).

Narciclasine (1,2,3,7-tetrahydroxy-8,9-methylendioxy-1,2,3,4-tetrahydrophena ntridone) is a natural substance with strong antimitotic effects on cells and potential antitumor activity. Its release form a hydrogel matrix was studied with the purpose of avoiding the concentration spikes of the parenteral administration. The matrix prepared by gamma ray polymerization of a mixture of 2-hydroxyethyl methacrylate (85%) and trimethylolpropane trimethacrylate (15%) was found to release narciclasine for several days, according to a diffusion controlled mechanism. In agreement with its antimitotic effect, narciclasine inhibited the growth rate of healthy mice, when the drug-loaded matrix was introduced subcutaneously. Antitumor effect was observed in an experimental model of Erlich ascitic tumor when low amounts of tumor cells were inoculated. No effect was observed at high concentrations of inoculum or towards solid tumors (Sarcoma 180). This behaviour was related to the rapid clearance of narciclasine from the body which prevented the reaching of sufficient therapeutical concentrations. A pharmacokinetic investigation carried out by an original method of assay demonstrated that narciclasine was accumulated in significant amounts in the kidney only and eliminated in urine with a half time of less than 20 min.

European Multicentre Evaluation of the ESAT 6660.

The analytical performance of the glucose analyser ESAT 6660 from Eppendorf was studied according to the ECCLS guidelines and partly according the SFBC protocol in a multicentre evaluation involving laboratories from three European countries. The glucose determination in serum and in haemolysate was studied. The following results were obtained. 1. The precision was as good as or better than the precision of the comparison instruments. The coefficients of variation were between 1.1 and 3.4% for the between-days imprecision and between 0.35 and 1.45% for the within-run imprecision experiment. 2. The recovery of control sera values compared with the hexokinase method was between 94.3 and 102.6%. 3. With patient specimens as good agreement was found between the results obtained with the ESAT 6660 and the different comparison instruments (ASTRA, Hitachi 737 and ACP 5040). 4. A drift effect of 1.1-2.3% occurred in 5 of 21 experiments, depending on the individual enzyme membrane. 5. Sample carry-over was not observed. 6. A linearity between 0.5 and 50 mmol/l was found, exceeding the manufacturer's claims. 7. Several different endogenous and exogenous interferences were investigated. No interfering effect was detected for endogenous substances. A positive interference was observed by ascorbic acid at a concentration above 350 mg/l. 8. The practicability of the instrument was judged as very good. It was considered as a disadvantage that the instrument is not capable of piercing sample lids. Also the numeration of samples is not very convenient.

Multi-centre evaluation of the urine test strip analyser Rapimat.

A multi-centre evaluation of the test strip analyser, Rapimat, was performed by four laboratories following the ECCLS 2nd draft guidelines for the evaluation of analysers in clinical chemistry. Using the Rapignost urine test strip with the test fields for bilirubin, urobilinogen, acetoacetate, ascorbic acid, glucose, protein, nitrite, pH and haemoglobin, the Rapimat was found to be analytically reliable in comparison with other, in most cases quantitative procedures. During the observation period of about 6 months no breakdown occurred in any laboratory. Interferences and sensitivity as discussed for the bilirubin and urobilinogen test field are more related to the test strip than to the instrument. Several improvements for further developments are suggested. This multi-centre study has shown that the ECCLS protocol is applicable to analytical procedures leading to discrete results.

Lipid bound sialic acid in cancer patients.

Serum lipid-bound sialic acid (LSA) was measured with a recently described procedure in 108 healthy subjects and in 138 patients with a variety of solid tumors and hematologic malignancies. At the time of serum sampling, 128 patients had active disease and 10 patients had no evidence of disease. LSA was elevated in 104 of 128 (81.2%) patients with active disease, while carcinoembryonic antigen, analyzed in 74, was elevated only in 21 (28.4%) (P less than 0.05). Sensitivity of the serum LSA test ranged from 66% for breast and gastrointestinal cancer to 92% for lung cancer. In patients with lung cancer, ovarian cancer or Hodgkin's disease, LSA was correlated with the extent of disease and it also proved to be useful in following the course of disease. Our preliminary data indicate that this test can be used as a monitor of tumor burden.

Pepsinogen Group I radioimmunoassay and total serum pepsinogen colorimetric determination: a comparative study in normal subjects and in peptic ulcer patients.

Pepsinogens, proteolytic enzymes produced by peptic cells of the stomach and discharged into the gastric lumen as well as into the blood have been divided into two groups: PG-I, originating from chief cells, and PG-II, mainly from antrum peptic cells. Both total serum pepsinogen (s-Pg) and PG-I have been separately reported as being significantly increased in gastric (GU) and duodenal ulcer (DU) patients and related to maximal acid output. In order to ascertain the relationship between s-Pg measured by means of the colorimetric Uete method, and PG-I determined by RIA method, these were assayed in 72 control subjects, 35 GU and 95 DU patients. s-Pg was found to be significantly increased both in GU and DU patients in comparison with control subjects. Likewise PG-I was significantly enhanced in GU and DU patients as compared with controls. A significant direct correlation between s-Pg and PG-I was found in all the subjects studied (r = 0.732).

Kinetic enzymatic determination of creatinine by a rapid semiautomated procedure.

The enzymatic method of creatinine determination, based on the transformation of creatinine to creatine by creatinine amido-hydrolase (EC 3.5.2.10) and subsequent evaluation of the creatinine, has been automated as a kinetic initial rate procedure. This was achieved by using a preincubation period under conditions that reduce the interference by accessory reactions to a very low and constant value, followed by a relatively high concentration of creatinine amido-hydrolase as a starter.

Role of trypsin/creatinine clearance ratio in the differential diagnosis of chronic pancreatic disease.

Trypsin/creatinine clearance ratio--a recently proposed screening test for pancreatic cancer--was assessed in 45 subjects (17 control subjects, 15 patients with pancreatic cancer, and 13 with chronic pancreatitis). A statistically significant increase of the ratio was detected not only in pancreatic cancer, but also in chronic calcifying pancreatitis. Thus, the previously reported clinical usefulness of the test in pancreatic cancer diagnosis was not substantiated by the present data. Although not fully investigated as yet, reasons for an abnormal ratio are probably independent of the neoplastic or inflammatory nature of the pancreatic disease. Science renal enzyme excretion (alpha-glucosidase, gamma-glutamyltranspeptidase, leucine aminopeptidase) was not found to be invariably elevated when trypsin/creatinine clearance ratio was increased, tubular damage cannot be assumed as constituting the only reason for an altered clearance ratio.

[Clinical importance of the determination of antithrombin III by the use of a chromogenic substrate, in cases of disseminated intravascular coagulation and of acute hepatic insufficiency]. / Rilevanza clinica della determinazione della antitrombina III mediante substrato cromogeno in casi di coagulazione intravascolare disseminata e di insufficienza epatica acuta.

Antithrombin III (AT III) an alpha 2 globulin produced by liver, is the most important plasmatic inhibitor of activated coagulation factors, that bind irreversibly to it with formation of inactive complexes. Therefore, when coagulation processes are activated in vivo, a decrease of AT III is presumably likely to occur. In the present research, AT III has been determined both as substance concentration, by radial immunodiffusion, and on the base of its activity on a chromogenic substrate (Chromozym) in patients with DIC before and after heparin therapy. Some patients with acute liver insufficiency have been similarly studied, because they not only have a deficient protein synthesis but also show phenomena of anticoagulative factors consumption. In all the patients, the AT III levels appeared decreased by both methods; the decrease of activity was comparatively much more intense and in a case no activity was even detectable.

[At II in liver diseases. Comparison between immunological determination and that with chromogenic substrate]. / L'AT III in patologia epatica. Confronto tra la determinazione immunologica e quella con substrato cromogeno.

Plasma antithrombin III activity determined as a heparinic cofactor, by means of chromogenic substrate (Cromozym TH) has been compared with protein concentration of AT III (radial immunodiffusion) in patients with a variety of liver conditions. Reference values for AT III were obtained from the plasma of 50 donors whose state of health was confirmed by simultaneous determination of 20 haematochemical parameters (SMAC-Technicon). The patients were classified according to clinical, laparobioptic and laboratory data and put through a series of clotting tests including PT, fibrinogen, FDP, Hepatoquick, Platelet count. In healthy donors, the activity and protein concentration of antithrombin III were interrelated, as they were even in cirrhotic patients (r = 0.77) both being markedly reduced; in chronic hepatitis, diminution with both methods was modest and correlation less apparent (r = 0.48).