Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Proviral tagging in E mu-myc transgenic mice lacking the Pim-1 proto-oncogene leads to compensatory activation of Pim-2.

The Pim-1 proto-oncogene is one of the most potent collaborators of the myc proto-oncogenes in inducing lymphomagenesis in mice. Contrary to the profound effects when overexpressed in vivo, Pim-1-deficient mice showed only subtle phenotypic alterations, which could indicate the presence of redundantly acting genes. In line with this, a PCR-based screen has led to the identification of a closely homologous gene, Pim-2. The X-linked Pim-2 gene is 53% identical to Pim-1 at the amino acid level and shares substrate preference and the usage of non-AUG initiation codons with Pim-1. We have used these data to test whether the strong synergistic interaction between Pim-1 and c-myc can be utilized to gain access to Pim-1 compensatory pathways. We reasoned that, upon proviral tagging in compound mutant mice (E mu-myc/Pim-1-/- mice), the selective advantage of cells carrying provirally activated genes, that act downstream from or parallel to Pim-1, would increase. We show here that this is the case. A dramatic increase (from 15 to 80%) was found in the frequency of proviral activation of the Pim-2 gene. These data show that the described strategy of 'complementation tagging' represents a powerful new tool to identify components of pathways involved in processes as complex as multistep tumorigenesis.     

The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P_GAP and P_AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P_GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P_AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.     

A method for estimating torque-vector directions of shoulder muscles using surface EMGs

In this study, a new method is proposed to estimate the torque-vector directions of each shoulder muscle. The method is based on a multiple regression model that reconstructs shoulder torque, which is calculated from the hand force and posture, from the surface EMG of many muscles recorded simultaneously. The torque-vector directions of eleven shoulder muscles of four subjects were obtained at up to 30 different arm postures with this method. The mean confidence interval (p < 0.05) of the estimated torque-vector direction of each subject was 7.7-10.6 degrees. The correlation coefficient between the measured shoulder torque and reconstructed shoulder torque was between 0.76-0.84. The results for majority of the muscles were in accordance with previous studies, and reasonable from the viewpoint of anatomy. The torque-vector directions of a muscle, which are estimated with this method, have more of a functional meaning than a pure anatomical or mechanical one. These indicate the direction of the shoulder torque accompanying the muscle activation for a normal shoulder action that involves the cooperative contraction of many muscles.     

Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions

The purification and characterization of an organic solvent tolerant, NADH-dependent medium-chain secondary alcohol dehydrogenase (denoted sec-ADH "A") from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2-propanol (up to 80% v/v). Thus, it is ideally suited for the preparative-scale enantioselective oxidation of sec-alcohol and the asymmetric reduction of ketones, using acetone and 2-propanol, respectively, as cosubstrates for cofactor-regeneration via a coupled-substrate approach. The homodimeric protein was found to bear tightly bound zinc and displayed a molecular mass of 38 kDa per subunit as determined by SDS gel electrophoresis. The optimal temperature ranged from 30-50 degrees C and the half-life at 50 degrees C was 35 h. In addition, excellent storage stability was found. The pH optimum for reduction is pH 6.5; pH 9.0 is preferred for oxidation. The enzyme followed a sequential reaction mechanism. The substrates are medium chain sec-alcohols or (omega-1)-ketones; primary alcohols or aldehydes are not accepted.     

The role of apoptosis in regulating hematopoietic stem cell numbers

The importance of apoptosis, in combination with proliferation, in maintaining stable populations has become increasingly clear in the last decade. Perturbation of either of these processes can have serious consequences, and result in a variety of disorders. Moreover, as the players and pathways gradually emerge, it turns out that there are strong connections in the regulation of cell cycle progression and apoptosis. Apoptosis, proliferation, and the disorders resulting from aberrant regulation have been studied in a variety of cell types and systems. Hematopoietic stem cells (HSC) are defined as primitive mesenchymal cells that are capable of both self-renewal and differentiation into the various cell lineages that constitute the functioning hematopoietic system. Many (but certainly not all) mature hematopoietic cells are relatively short-lived, sometimes with a half-life in the order of days. Homeostasis requires the production of 10⁸ (mouse) to 10¹¹ (human) cells each day. All of these cells are ultimately derived from HSC that mostly reside in the bone marrow in adult mammals. The study of the regulation of HSC numbers has focussed mainly on the choice between self-renewal and differentiation, symmetric and asymmetric cell divisions. Recently, however, it has been directly demonstrated that apoptosis plays an important role in the regulation of hematopoietic stem cells in vivo.     

Efficient Solar‐Driven Water Oxidation over Perovskite‐Type BaNbO2N Photoanodes Absorbing Visible Light up to 740 nm

Photoelectrochemical water splitting using semiconductors absorbing a wide range of visible light is a potentially attractive means of harvesting large portions of the solar spectrum. However, this is also very challenging because narrowing the semiconductor band gap lowers the driving force for photoreactions. Herein, a highly active perovskite BaNbO₂N exhibiting photoexcitation up to 740 nm for water oxidation is reported. The synthesis route, consisting of moderate nitridation and subsequent annealing in inert Ar flow, enhances the crystallinity of the BaNbO₂N surface without inducing the reduction of the Nb species. As a result, a particulate BaNbO₂N photoanode exhibits a photocurrent of 5.2 mA cm^?2 at 1.23 V_RHE under simulated solar irradiation, which is the highest yet reported for an oxynitride responsive at wavelengths above 600 nm. Suppressing the reduction of B‐site cations during the synthesis of perovskite AB(O,N)₃, which otherwise results in surface defects or impurities, is critical for achieving high water oxidation activity.