A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.     

Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

Mitotic chiasmata in human diplochromosomes

Summary Three placental tissue cultures of spontaneous human abortions showed an unusually high frequency of metaphases with diplochromosomes. In 62 such cells, nine configurations were interpreted as mitotic chiasmata between the two sister chromosomes of a diplochromosome. One U-type exchange between two sister chromosomes was also found. This differs significantly from the 1:1 ratio of adjacent and alternate exchanges in translocations, thus supporting the idea that mitotic chiasmata are in principle different from chromatid translocations. The hypothesis is put forward that the frequency of homologous exchanges is determined by the intimacy of pairing which ranges from meiotic pairing through sister chromatid association, through sister chromosome association in diplochromosomes to accidental pairing of homologous regions in diploid cells.     

Structure and Barr body formation of an Xp + chromosome with two inactivation centers.

A patients with seizures, Von Willebrand disease, and symptoms of Turner syndrome was a chromosomal mosaic. In blood culture (1974), 56% of the cells were 45, X 33% 46, XXp+ and 11% 47,XXp + Xp +; in the skin, no cells with 47 chromosomes were found. Presumably the Xp + chromosome arose through a break in the Q-banded dark region next to the centromere on Xp to which an Xq had been attached. The abnormal X was late-labeling and formed a larger than normal Barr body. Of the chromatin-positive fibroblasts, 18.2% showed bipartite Barr bodies, which agrees with the hypothesis that the X inactivation center lies on the proximal part of the Xq. On the basis of the structure and behavior of the bipartite bodies in the present patient, as compared to those formed by other chromosomes with two presumed inactivation centers, we propose that the dark region next to the centromere of Xp remains active in the inactive X. In cells with 45,X and 46,XY, this region has the same relative size, whereas it is significantly shorter in the active X of three females, including the present patient, with one abnormal X. We propose that this region on the active X reveals different states of activity, as reflected in its length, depending on how many other X chromosomes are in the cell.     

How can storage time and temperature affect enzymic activities in urines?

We measured a tubular brush-border enzyme (alanine aminopeptidase, AAP) and a lysosomal hydrolase (N-acetyl-beta-D-glucosaminidase, NAG) in morning urines from 15 healthy normal subjects to check if different storage times and temperatures could modify enzyme concentrations. Short-term (24 h) storage time at room temperature or 4 degrees C does not affect AAP and NAG activities. Both enzymes are well preserved at -70 degrees C. AAP dramatically falls after 1 month at -20 degrees C, lowering to about 8% of the initial value after only 4 days of storage. On the contrary, NAG is well preserved at these storage conditions. Centrifugation has revealed not critical for measurement of these two enzymes.     

Automated measurement of lactate dehydrogenase, alkaline phosphatase and gamma-glutamyltransferase in urines: an alternative to the manual procedure.

A sensitive and precise automated assay of urinary lactate dehydrogenase (EC 1.1.1.27), alkaline phosphatase (EC 3.1.3.1) and gamma-glutamyltransferase (EC 2.3.2.2) is described. For this purpose, we used a BM/Hitachi System 704 model and reagents for automated analysis of serum enzymes from Boehringer Mannheim. However, the schedules of enzyme chemistry parameters recorded by the autoanalyzer and the spectrophotometric calibration are reprogrammed to meet requirements deriving from urine adoption and to optimize the enzyme assay in this unusual medium.     

Localization of the nucleolar organizer by computer-aided analysis of a variant no. 21 in a human isolate

Summary A variant chromosome no. 21 consisting of two stalks and two satellites in tandem was detected during a survey of a human isolate. The variant segregated in three generations of a large kindred. One male had the variant no. 21, a metacentric Y, and a 47, XXY complement; however, no other evidence of chromosomal nondisjunction was found. Computer-aided analysis of sequentially stained variant no. 21 chromosomes indicated that silver-stained material corresponded to the proximal stalk region (as defined defined by Giemsa). These data support the hypothesis that human nucleolar organizers are localized to the stalks of acrocentric chromosomes.     

Proteome profile of human urine with two-dimensional liquid phase fractionation

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.     

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.