The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.     

Osteogenesis imperfecta: a heterogeneous morphologic phenotype in cultured dermal fibroblasts

Summary Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n=6) and OI cell strains (n=10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300xmagnification). Our results show that: (i) OI fibroblasts take longer (11–27 days, mean 20 days) than control cells (10–19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87x10⁶ cells/P60, range 0.3–1.6x10⁶) at stationary phase relative to control cells (2.2x10⁶ cells/P60, range 1.7–2.6x10⁶; F_4,77=56.1, p<0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width: length ratio) was also abnormal in OI cells. (F_4,730=37.6, p<0.01), types I and II OI cells have significantly increased ratios (p<0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.Publication No. 84013 from the Montreal Children's Hospital Research Institute     

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 μg/ml. Recoveries varied from 91 to 10% with coefficients of variation ranging from 0.387 to 7.95%.     

Survival of Escherichia coli in the environment: fundamental and public health aspects

In this review, our current understanding of the species Escherichia coli and its persistence in the open environment is examined. E. coli consists of six different subgroups, which are separable by genomic analyses. Strains within each subgroup occupy various ecological niches, and can be broadly characterized by either commensalistic or different pathogenic behaviour. In relevant cases, genomic islands can be pinpointed that underpin the behaviour. Thus, genomic islands of, on the one hand, broad environmental significance, and, on the other hand, virulence, are highlighted in the context of E. coli survival in its niches. A focus is further placed on experimental studies on the survival of the different types of E. coli in soil, manure and water. Overall, the data suggest that E. coli can persist, for varying periods of time, in such terrestrial and aquatic habitats. In particular, the considerable persistence of the pathogenic E. coli O157:H7 is of importance, as its acid tolerance may be expected to confer a fitness asset in the more acidic environments. In this context, the extent to which E. coli interacts with its human/animal host and the organism''s survivability in natural environments are compared. In addition, the effect of the diversity and community structure of the indigenous microbiota on the fate of invading E. coli populations in the open environment is discussed. Such a relationship is of importance to our knowledge of both public and environmental health.     

Possible regulation of pyruvate carboxylase fromThiobacillus novellus by hydroxypyruvate

Aspartate, glutamate, or dicarboxylic acids did not inhibit the activity of a highly purified but not homogeneous preparation of pyruvate carboxylase fromThiobacillus novellus. The only effective inhibitors were end-products of the reaction and to a lesser degree hydroxypyruvate. The latter has not been shown previously to regulate the enzyme's activity. Lineweaver-Burk plots revealed that it was uncompetitive with respect to acetyl CoA with a K_ii of 3.6 mM, and noncompetitive with respect to bicarbonate, magnesium ATP, and pyruvate with respective K_ii values of 7.1, 5.5, and 6.47 mM. The corresponding K_is values were 7.02, 5.4, and 4.25 mM. A mathematical model is presented that supports the findings.     

From the Schnauzenorgan to the back: Morphological comparison of mormyromast electroreceptor organs at different skin regions of Gnathonemus petersii

The nocturnally active weakly electric fish Gnathonemus petersii is known to employ active electrolocation for the detection of objects and for orientation in its environment. The fish emits pulse‐type electric signals with an electric organ and perceives these signals with more than 3,000 epidermal electroreceptor organs, the mormyromasts, which are distributed over the animal's skin surface. In this study, we measured the metric dimensions of the mormyromasts from different body regions to find structural and functional specialization of the various body parts. We focused on the two foveal regions of G. petersii, which are located at the elongated and movable chin (the Schnauzenorgan; SO) and at the nasal region (NR), the skin region between the mouth and the nares. These two foveal regions were compared to the dorsal part (back) of the fish, which contains typical nonfoveal mormyromasts. While the gross anatomy of the mormyromasts from all skin regions is similar, the metric dimensions of the main substructures differed. The mormyromasts at the SO are the smallest and contain the smallest receptor cells. In addition, the number of receptor cells per organ is lowest at the SO. In contrast, at the back the biggest receptor organs with the highest amount of receptor cells per organ occur. The mormyromasts at the NR are in several respects intermediate between those from the back and the SO. However, mormyromasts at the NR are longer than those at all other skin regions, the canal leading from the receptor pore to the inner chambers were the longest and the overlaying epidermal layers are the thickest. These results show that mormyromasts and the epidermis they are embedded in at both foveal regions differ specifically from those found on the rest of the body. The morphological specializations lead to functional specialization of the two foveae. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.