Reliability of copper reduction methods for quantitation of glucosuria

In the present study we evaluated the reliability of the "2-drop" Clinitest method in determining the 24-h glucose spill of a diabetic patient. The urine glucose content over the 33 day study period was measured with a Beckman glucose analyzer that employed a glucose oxidase method. There was no statistical difference between the glucose content determined by the two methods. There was a mean error of +4.4 +/- 3.6% between the values obtained with the "2-drop" method and values obtained with glucose analyzer. The mean coefficient of variation for determining the glucose content of urine with the "2 drop" method was 19%. In a group of 10 normal volunteers we found that there was a falsely elevated glucose value in urines with osmolality of less than 295 mosm/kg when the "5-drop" method was used. The "2-drop" Clinitest and the Beckman analyzer could accurately determine glucose concentration even in these dilute urines. The "2-drop" Clinitest method is a useful and reasonably reliable method for evaluationg 24-h glucosuria.     

The clinical utility of viral quantitation using molecular methods

BACKGROUND: The quantitation of viral nucleic acids in biological fluids has become increasingly desirable over the past several years. To this end, a number of quantitative molecular procedures have been developed. OBJECTIVES: The objective was to review the current literature on the molecular techniques used in the quantitation of viral nucleic acids and to assess the appropriateness of these methods for clinical use. RESULTS: Assays involving both target and signal amplification are now available for the accurate and precise quantitation of viral burden in infected patients. These methods include quantitative polymerase chain reaction (PCR), branched chain signal amplification (bDNA), nucleic acid sequence-based amplification (NASBA) and the SHARP signal and hybrid capture systems. Our understanding of the natural history and pathogenesis of viruses such as the human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) may be greatly facilitated by accurate determinations of viral and infected cell burden. Quantitation of viral load in infected individuals may also be useful to assess disease progression, monitor the efficacy of therapy and to predict treatment failure and the emergence of drug-resistant viruses. CONCLUSION: Precise, accurate and reproducible quantitation of viral load is now feasible. Molecular assays for viral quantitation should have a considerable impact on medical research and clinical care.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

Evaluation of the methods for calculating the concentration-dependent diffusivity in binary systems

Boltzmann and Matano developed a procedure for the solution to Fick’s second law when the diffusivity is a function of concentration. The procedure requires the determination of the so-called Matano interface. The accuracy of the resulting solution depends heavily on the precise location of the Matano interface, the determination of which is laborious and often inaccurate. Three alternative procedures by Sauer and Freise, Wagner, and den Broeder, modifications to the original Boltzmann-Matano (B-M) method, were developed such that the diffusivity can be calculated without having to determine the location of the Matano interface. However, none of these derivations quantifies the extent to which the modified methods arrive at the same result as that obtained from the standard B-M analysis. This article serves to apply the B-M method, and the modification suggested by den Broeder, to various analytical concentration profiles containing different degrees of noise and to compare the results quantitatively. In addition to these analytical functions, concentration profiles obtained from interdiffusion experiments were studied. The two methods are shown to be equivalent in terms of the accuracy of the result. The advantages or disadvantages of one method over the other are illustrated with examples.     

Immunohistochemical quantitation for extracellular matrix proteins in rats with glomerulonephritis induced by monoclonal anti-Thy-1.1 antibody

During clot retraction, platelets interact with fibrin resulting in marked reduction of clot volume. Altered fibrin structure has been reported to affect clot retraction as measured by serum expression. This study was performed to test whether such altered retraction was the result of increased resistance to network collapse or due to decreased force development by platelets. Altered fibrin structure was documented as variation of fibre mass/length ratios (mu) and shifts in clot elastic modulus. The force developed by platelets during clotting was measured directly. Increasing the fibrinogen concentration led to thinner fibre formation (decreased mu), and a linear increase in gel elastic modulus. Over a fibrinogen concentration range of 100 to 400 mg/dl, force development was minimally affected. Force development and clot elastic modulus increased in a linear fashion with increasing platelet concentration. Increasing the calcium concentration from 5 to 20 mM caused a 160% increase in fibrin fibre size (mu), and a 52% decline in clot modulus. Force developed at 1200 s declined by 17%. At 15 mg/ml, dextran and hydroxyethyl starch (HES) also increased mu, and decreased clot modulus; however, both agents markedly reduced force development. Increasing ionic strength or the addition of IgG decreased mu and increased gel elastic modulus. Force development increased modestly with increased ionic strength, did not change with addition of IgG in saline and declined with addition of IgG in maltose. This study indicates that force development is primarily dependent on platelet function while clot modulus depends on both fibrin structure and platelet function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of in vitro precipitation methods

As CD44 is believed to be a homing receptor involved in lymphoid trafficking and inflammatory responses, it is expected to be closely linked to transplant rejection. In this study, the expression of CD44 during liver transplant rejection was compared with the expression of lymphocyte-function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), which play an essential role in cell interactions and the initiation of immune responses. Male Brown Norway (BN) and Lewis (LEW) rats were used as donors and recipients, respectively. Orthotopic liver transplantation (OLTX) was done using the cuff technique of Kamada and Calne. Animals were killed on days 3, 5, and 7 after OLTX, and a piece of tissue from each of the liver grafts was obtained. Immunohistochemical staining was used to investigate the expression of CD44, ICAM-1, and LFA-1. CD44 was strongly expressed in portal areas of the rejected liver, and LFA-1 and ICAM-1 were expressed mainly on sinusoids and hepatocytes. These findings indicate that CD44 is closely involved in lymphocyte infiltration, which is dominant in portal areas, and that lymphocyte infiltration during the rejection process may involve a homing mechanism.     

The use of electrophoretic methods for determining modified proteins in diabetic patients

Modified proteins were determined by isoelectric focusing in borate-polyol system with subsequent colorimetry (micromethod) and electrophoresis of blood serum on paper with subsequent TCA-ethanol treatment. Increased levels of glycated hemoglobin and modified albumin and changed light absorbance of glycated albumin were detected. The levels of glycated hemoglobin assessed by the micromethod and colorimetry without calibration did not correlate.     

Evaluation of the Nuclisens HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 RNA levels in plasma

Nuclisens HIV-1 QT is a new version of the NASBA HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma. The specificity of this assay was 100% in one laboratory and 99%-with nonrepeatability of the initial false positive-in another. The test was linear between 2.0 and 6.0 log RNA copies per ml. According to the input HIV-1 RNA concentration, accuracy varied from -0.11 to +0.10 log RNA copy per ml and precision varied from 0.66 to 0.14 log RNA copy per ml. Reproducibility decreased when the HIV-1 RNA level was near the lower limit of quantitation of the test. HIV-1 RNA could be quantitated by Nuclisens HIV-1 QT in 36% (laboratory 1) and 24% (laboratory 2) of clinical samples with HIV-1 RNA levels lower than the lower limit of quantitation by NASBA HIV-1 QT. Nuclisens HIV-1 QT was not suitable for measurement of RNA from clade G and group O HIV-1 strains.     

Evaluation of methods of sperm preparation for human in vitro fertilization

Congenital absence of useful ilio-caval venous segment is a very infrequent congenital anomaly and makes unfit grafting of a kidney transplant in iliac fossa. We report the case of a 18 years old male affected by this abnormality who was transplanted in intraabdominal situation. We review technical alternatives offered by the literature.     

Evaluation and comparison of three mobilization methods for the collection of granulocytes

BACKGROUND: Cancer chemotherapeutic regimens have become more potent and myeloablative. As a consequence, morbidity and mortality due to opportunistic infections have become a major challenge. The provision of adequate doses of viable granulocytes has thus become an important approach for circumventing the problem. A schedule for collecting therapeutic numbers of cells with minimal donor toxicity has yet to be established. STUDY DESIGN AND METHODS: An investigation of three mobilization schedules for the collection of granulocytes for transfusion--granulocyte-colony-stimulating factor (G-CSF) 5 micrograms per kg daily; G-CSF 5 micrograms per kg every other day, and prednisone 60 mg given orally (20 mg doses at 17 hours, 12 hours, and 2 hours before the collection). RESULTS: A total of 464 apheresis procedures involving 163 healthy donors were analyzed. Prednisone caused a small increase in the white cell (WBC) counts over the collection days, while G-CSF every other day and daily schedules improved WBC counts to 145 and 160 percent, respectively (p = 0.004). Similarly, administration of G-CSF daily and every other day mobilized higher yields of granulocytes over the collection days, compared to the prednisone schedule (170% and 180% vs. 105%; p = 0.02). CONCLUSION: Compared with prednisone, higher WBC yields were achieved by G-CSF stimulation; G-CSF given every other day is as effective as daily G-CSF administration for the recruitment of granulocytes, which makes the mobilization procedure more cost-effective.     

Functional analysis of conserved cysteines in heparan sulfate N-deacetylase-N-sulfotransferases

N-Deacetylase-N-sulfotransferases (NDANST) catalyze the two initial modifications of the polysaccharide precursor in the biosynthesis of heparin and heparan sulfate. These modifications are the gating steps in establishing growth factor protein-binding domains of these glycosaminoglycans. We have undertaken a structure-activity analysis of the 841-amino acid Golgi-luminal portion of the rat liver NDANST to localize the two enzymatic functions. Each activity can be assayed in vitro independently of the other when provided with the appropriate substrate, and N-ethylmaleimide treatment selectively inactivates the deacetylase activity. In this study, dithiothreitol treatment of the rat liver NDANST was shown to inactivate the sulfotransferase function, while stimulating deacetylase activity 2-3-fold over the native protein. Site-directed mutagenesis of the eight cysteine (Cys) residues in the rat liver NDANST that are conserved in the mouse mastocytoma protein produced three important findings regarding the localization of each enzymatic function: 1) derivatization of Cys486 with N-ethylmaleimide resulted in total inactivation of the deacetylase activity based on steric hindrance of the active site (this residue was shown not to be involved in enzymatic catalysis), 2) substitution of either Cys159 or Cys486 with alanine resulted in enhanced activity of the deacetylase to the level obtained by dithiothreitol treatment, and 3) alanine substitution of Cys818 or Cys828 completely inactivated the sulfotransferase activity, while substitution of Cys586 or Cys601 resulted in a 90% loss in activity. These findings suggest that the two enzymatic domains within the NDANST localize to different portions of the protein, with two disulfide pairs toward the COOH-terminal half of the protein necessary for the sulfotransferase activity, and Cys residues within the NH2-terminal half influencing or located near the active site of the deacetylase functionality.     

Comparison of methods for separating proteins using bisalbuminemia as a model

Sera from bisalbuminemic chicken-turkey hybrids contain two albumins in equal amounts. These are observed as inherited electrophoretic variants and originate from the respective chicken and turkey parents. Sera from the hybrid birds served as a model system by which fractionating and indentification procedures for evaluating serum albumin variants were compared. The two albumins in the hybrid were isolated with preparative polyacrylamide gel electrophoresis (PAGE) and starch block preparative electrophoresis. Isoelectric focusing of the hybrid albumins resulted in the isolation of the turkey albumin. Interference of ampholines prevented the complete isolation of the chicken albumin. The two albumins in the hybrid have identical molecular weights and cannot be identified by sedimentation coefficient, gel filtration behavior, or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the close relatedness the chicken and turkey albumins in the hybrid cross reacted with rabbit anti-hybrid serum as well as with rabbit anti-chicken anti-turkey sera.     

Evaluation of the ultrasensitive Roche Amplicor HIV-1 monitor assay for quantitation of human immunodeficiency virus type 1 RNA

The ultrasensitive Amplicor HIV-1 Monitor test (Roche Diagnostic Systems) was evaluated for precision, linearity, and sensitivity and was compared to the standard Amplicor assay. The ultrasensitive assay reliably quantified samples in the range from 50 to 50,000 human immunodeficiency virus type 1 RNA copies/ml with acceptable correlation with the standard Amplicor test.     

Two replica blotting methods for fast immunological analysis of common proteins in two-dimensional electrophoresis

Knowledge of the photon spectrum of a radiotherapy beam is often needed for three-dimensional (3-D) dose calculations using Monte Carlo methods and/or algorithms employing energy deposition kernels. Direct measurement of the x-ray energy fluence spectrum is not feasible for the high-energy photon beams used clinically. In this paper, the spectrum is extracted from basic beam data that are readily obtained for a clinical beam. We describe the photon spectrum using just two parameters. One parameter, which determines the high-energy part of the spectrum, is obtained using the measured dose in the buildup region for a small field, where electron contamination of the beam can be neglected. The other parameter is extracted from the photon beam attenuation in water. The results compare favorably to spectra generated from Monte Carlo simulations.     

Evaluation of recommended methods for radioisotopes red cell survival studies

Mean red cell life-span in normal subjects and in patients with various hematological disorders was examined with 51Cr and DF32P. The results with 51Cr were corrected for 51Cr elution using correction factors. The results by the two methods agreed fairly well with each other. Elution rate in various hematological disorders was 2.3% per day or less except for the patients with extracorpuscular hemolytic agents such as autoimmune hemolytic anemia or congestive splenomegaly. It is concluded that estimates of mean red cell life-span by corrected 51Cr method are more useful and sufficient than uncorrected 51Cr or DF32P method in general hematological disorders.     

Evaluation of radioimmunological methods for assay of plasma and urinary aldosterone

Two radioimmunological methods for assay of plasma and urinary aldosterone were carefully evaluated. In the plasma method a radioimmunoassay is preceded by chromatography on a Sephadex LH-20 column. The method for urine includes a preextraction, hydrolysis of the acid-labile conjugates of aldosterone, and a radioimmunoassay. Both methods fulfill the criteria of reliability and are suitable for both routine and demanding research assays. The plasma method, using columns of double length, is also applicable to analysis of aldosterone in plasma of newborn children, and pregnant females and in cord plasma. The concentration of plasma aldosterone in healthy subjects on an ad lib salt diet was 162 +/- 93 (S.D.) pmol/1 in the supine position and 312 +/- 217 (S.D.) pmol/1 upright. The urinary excretion of aldosterone in healthy subjects was 28.3 +/- 16.7 (S.D.) nmol/24 h.     

Anti-lipid A antibodies in childhood arthritis: methods of immobilization affect quantitation and crossreactivity measured by ELISA

OBJECTIVE: To find an optimal method to study antibodies reactive with monophosphoryl lipid A characteristic of oligoarticular arthritis in children. METHODS: ELISA using 3 different methods of immobilization were compared, in (1) HCO3 buffer, pH 9.6; (2) HCl, pH 2.0; and (3) methanol. Competitive inhibition studies were carried out to quantitate relative avidity of cross reactions with suspected autoantigens. RESULTS: Sera from healthy children reacted significantly more strongly with monophosphoryl lipid A after immobilization in acid or in methanol than in a basic buffer. Sera from children with oligoarticular arthritis reacted more strongly than normal sera with the basic buffer method and even more strongly with the methanol method, but were not distinguishable from normal sera with the acid method. Results with individual oligoarticular sera correlated from method to method, but results with normal sera did not. Collagen types I and II, cardiolipin, and denatured DNA can block the anti-monophosphoryl lipid A reactivity to varying degrees on plates prepared with basic buffer, but only collagen type I and DNA block reactivity on plates prepared with methanol. CONCLUSION: The epitope on monophosphoryl lipid A recognized by oligoarticular sera is differentially affected by the method of immobilization. The crossreactivity of the anti-monophosphoryl lipid A antibody in this disease is confirmed.     

Influence of dipeptide derivatives of S-substituted cysteines on the time of fibrinolysis

Amino acids containing sulphur, dipeptide derivatives of methionine and S-substituted derivatives of cysteine are potent antifibrinolytic agents. The structural moiety of the substances responsible for the effect on the clot formation is not known. Present study was undertaken in order to evaluate the effect of some analogues of dipeptides containing S-substituted derivatives of cysteine with the formula A-Cys(S-X)-Y (where A-amino acid, X-benzyl, butyl, hexyl, nonyl and Y-OH or OMe) on clot dissolution under the antifibrinolytic test conditions. It has been found that dipeptide derivatives of S-substituted cysteine (except benzyl derivative) at low concentration evoke antifibrynolytic activity, while at high concentration they prevent clot formation. The results suggest that antifibrinolytic activity of tested compounds at low concentration may be due to the formation of antifibrinolitycally active conformation, while high concentration overcome the effect.