Quantitation of antibody uptake on A group erythrocytes using immunoautoradiography and monoclonal IgM anti-A

The number of antibody molecules on individual erythrocytes was counted in A1, A2, A3 B and A group individuals using immunoautoradiography (IAR) and monoclonal IgM anti-A1. Quantitation was also done for A group pregnant women. The number of antibody molecules on different red cells of an individual varied widely. Gross variations were also noted in cells of different individuals from one and the same group. The mean values of the uptake of the number of antibody molecules showed the following range A1 greater than A2 greater than Ax greater than A3B. When compared to the average for total A1 adults, red cells of pregnant women and newborn infants showed a 10.7% and 19.7% reduction respectively, in antibody uptake. The mean number of antibody molecules per A1 adult red cells was 5.6 +/- 3 X 10(4), while A2 had 0.85 +/- 0.35 X 10(4) molecules, thus showing a significant quantitative variation.     

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

 Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its K_M was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn²⁺ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities. Accepted: 28 August 1995     

Auer-rod positive myelocytic leukemia cells in diffusion chambers: differentiation along the eosinophilic pathway

Auer-rod positive acute myelocytic leukemia (AML) blasts from a 33-year-old patient were cultured in diffusion chambers (DC) in order to test their differentiation potential. The cells were labeled with anti-human granulocyte anti-serum known to be negative for eosinophils, and evaluated using the unlabeled peroxidase-antiperoxidase (PAP) method. Parallel to a decline in the number of leukemic blasts there was an increase of up to 86% in the number of granulocytic cells belonging to the eosinophilic series. Auer-rod bodies were found in the eosinophilic cells even after 20 days in culture. Staining with anti-granulocyte antiserum failed to demonstrate positive cells at any time during DC culture. Based on the negative reaction with the anti-granulocytic antibodies already at an early stage of development evidence is provided for the existence of a progenitor cell exclusively committed to the eosinophilic pathway.     

Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae,inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C₂H₄·mg protein^-1·h^-1 to 400 nmol C₂H₄ by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O₂, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce¹⁴CO₂ from [U-¹⁴C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 mol in 24 h by 2·10¹² cells) were metabolized to¹⁴CO₂, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O₂ in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.     

Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.