Galactokinase Mutants of Chinese Hamster Somatic Cells Resistant to 2-Deoxygalactose

The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase(EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & kmax of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possible a missence mutation, in the galactokinase gene.- A simple diagnostic test for juvenile cataract is proposed.     

Hemocyanin–antibody labeling of rhodopsin in mouse retina for a scanning electron microscope study

To reveal the presence of rhodopsin on the surface of the mouse retina, a scanning electron microscope study of the immunolabeling of rhodopsin was attempted. The glutaraldehyde-fixed mouse retina was treated first with rabbit antibodies specific against bovine rhodopsin and then with hemocynin-labeled goat antibodies specific against rabbit antibody. The distribution of hemocyanin label on mouse retina and the technique used for labeling are discussed.     

The effect of the elongation of the proximal aorta on the estimation of the aortic wall distensibility

The compliance of the proximal aortic wall is a major determinant of cardiac afterload. Aortic compliance is often estimated based on cross-sectional area changes over the pulse pressure, under the assumption of a negligible longitudinal stretch during the pulse. However, the proximal aorta is subjected to significant axial stretch during cardiac contraction. In the present study, we sought to evaluate the importance of axial stretch on compliance estimation by undertaking both an in silico and an in vivo approach. In the computational analysis, we developed a 3-D finite element model of the proximal aorta and investigated the discrepancy between the actual wall compliance to the value estimated after neglecting the longitudinal stretch of the aorta. A parameter sensitivity analysis was further conducted to show how increased material stiffness and increased aortic root motion might amplify the estimation errors (discrepancies between actual and estimated distensibility ranging from − 20 to − 62%). Axial and circumferential aortic deformation during ventricular contraction was also evaluated in vivo based on MR images of the aorta of 3 healthy young volunteers. The in vivo results were in good qualitative agreement with the computational analysis (underestimation errors ranging from − 26 to − 44%, with increased errors reflecting higher aortic root displacement). Both the in silico and in vivo findings suggest that neglecting the longitudinal strain during contraction might lead to severe underestimation of local aortic compliance, particularly in the case of women who tend to have higher aortic root motion or in subjects with stiff aortas.

     

Synthesis of Non-Natural Pyrimidine Nucleosides

Abstract

Two new non-natural nucleosides bearing an amide (8) or an amidine (9) function have been synthesized. Their properties and the geometry of the exocyclic double bond have been studied.     

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD⁺ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.     

Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida,Aspergillus and mucormycetes

Background aimsInvasive fungal infections, in particular, infections caused by Candida, Aspergillus and mucormycetes, are a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive transfer of donor-derived anti-fungal T cells shows promise to restore immunity and to offer a cure. Because T cells recognize only specific epitopes, the low rate of patients in which the causal fungal pathogen can be identified and the considerable number of patients with co-infection with several genera or species of fungi significantly limit the application of adoptive immunotherapy.MethodsUsing the interferon-γ secretion assay, we isolated multi-specific human anti-fungal T cells after simultaneous stimulation with cellular extracts of Aspergillus fumigatus, Candida albicans and Rhizopus oryzae. Cells were phenotypically and functionally characterized by flow cytometry.ResultsOf a total of 1.1 × 10⁹ peripheral blood mononuclear cells, a median number of 5.2 × 10⁷ CD3⁺CD4⁺ T cells was generated within 12 days. This cell population consisted of activated memory T_H1 cells and reproducibly responded to a multitude of Aspergillus spp., Candida spp. and mucormycetes with interferon-γ production. On re-stimulation, the generated T cells proliferated and enhanced anti-fungal activity of phagocytes and showed reduced alloreactivity compared with the original cell fraction.ConclusionsOur rapid and simple method of simultaneously generating functionally active multi-specific T cells that recognize a wide variety of medically relevant fungi may form the basis for future clinical trials investigating adoptive immunotherapy in allogeneic hematopoietic stem cell transplantation recipients with invasive fungal infection.     

Retinal Vascular Caliber Is Associated with Cardiovascular Biomarkers of Oxidative Stress and Inflammation: The POLA Study

Purpose

Retinal vascular caliber has been linked with increased cardiovascular risk and is predictive of cardiovascular pathology, including stroke and coronary heart disease. Oxidative stress, as well as inflammatory mechanisms, plays a major role in the pathogenesis and progression of atherosclerosis, plaque rupture and vascular thrombotic propensity. The purpose of this study is to explore the relationship between retinal vascular calibers and biomarkers of oxidative stress and inflammation, in subjects free of cardiovascular pathology.

Patients and Methods

Cross-sectional analysis from a community-dwelling cohort comprising 1224 individuals aged 60 years and over, without a history of coronary or peripheral artery disease or stroke. Retinal vascular caliber was measured from fundus photographs using semi-automated standardized imaging software. Oxidative stress was evaluated using plasma superoxide dismutase 2 and glutathione peroxidase (GPx-3) activities, and inflammatory state was assessed using plasma high sensitivity C-reactive protein (hsCRP) and orosomucoid.

Results

In a multivariate model controlling for cardiovascular risk factors, larger retinal arteriolar caliber was independently related to higher level of GPx-3 activity (p = 0.003) whereas larger venular caliber was associated with higher levels of hsCRP (p = 0.0001) and orosomucoid (p = 0.01).

Conclusion

In the present study, biomarkers of oxidative stress regulation and inflammation were independently associated with retinal vascular calibers. This suggests that an assessment of retinal vessels may offer early and non-invasive detection of subclinical vascular pathology.     

Peculiar tooth renewal in a Jurassic ray-finned fish (Lepisosteiformes, †Scheenstia sp.)

Tooth replacement in vertebrates is extremely diverse, and its study in extinct taxa gives insights into the evolution of the different dental renewal modes. Based on μ-CT scans of a left lower jaw of the extinct fish †Scheenstia (Actinopterygii, Lepisosteiformes), we describe in detail a peculiar tooth replacement mode that is, as far as we could ascertain from the literature, unique among vertebrates. The formation of the replacement teeth comprises a 180° rotation of their acrodin cap that occurs intraosseously within bony crypts, and their setting up appears to be synchronous. We propose a model for the dental renewal process and identify complementary anatomical features visible in the tomography such as the junction between the different tooth-bearing bones (prearticular–coronoid and dentary), as well as cavities corresponding to intraosseous crypts, nervous and/or vascular canals. The location of the cavities and their subsequent identification (e.g. Meckel's cavity, mandibular sensory canal) help us to identify the function of pores visible on the bone surface and understand their relation to internal anatomical features. Finally, recognition of this tooth replacement mode raises the question of whether it is specific to †Scheenstia or related to a particular dentition type and thus potentially occurs in other lineages.     

Middle Pleistocene ecology and Neanderthal subsistence: Insights from stable isotope analyses in Payre (Ardèche,southeastern France)

The Middle Palaeolithic site of Payre in southeastern France yields abundant archaeological material associated with fossil hominid remains. With its long sequence of Middle Pleistocene deposits, Payre is a key site to study the Middle Palaeolithic chronology of this region. This study is the first to investigate carbon and oxygen isotope contents of Neanderthal tooth enamel bioapatite, together with a wide range of herbivorous and carnivorous species. The aim is to contribute to the understanding of hunting behaviour, resource partitioning, diet and habitat use of animals and Neanderthals through a palaeoecological reconstruction.     

Development of an analytical approach for identification and quantification of 5-α-androst-16-en-3-one in human milk

A method was developed for the quantification of 5-α-androst-16-en-3-one in human breast milk based on application of a stable isotope dilution assay using 5α-androst-16-en-3-one-6, 6-d₂. The procedure includes extraction of the human milk by hexane with subsequent clean-up of the obtained extract by gel permeation and silica gel column chromatography. The extracted samples were analyzed by gas chromatography–mass spectrometry. Using this method 5-α-androst-16-en-3-one could be identified and for the first time quantified in a concentration range of 26–155 ng/kg in human milk.