Qualitative and quantitative observations on the structure of the Schwann cells in myelinated fibres

Various morphological features of the Schwann cells of myelinated fibres in the lizard thoracic spinal roots were studied, and, when possible, quantified using morphometric methods. About 0.8% of the Schwann cells are binucleate and some display clusters of microvilli along the internodes. The percentages of the cytoplasmic area of the Schwann cell occupied by the following cytoplasmic components were determined: mitochondria, Golgi apparatus, granular endoplasmic reticulum, smooth endoplasmic reticulum, multivesicular bodies, dense bodies, autophagic vacuoles, peroxisome-like bodies, lipofuscin granules and lipid droplets. Linear relationships were found between the sectional areas of the mitochondria and granular endoplasmic reticulum of the Schwann cell and both the length of the profile of the Schwann cell plasma membrane and the size of the related axon. The results obtained are compatible both with the hypothesis that the mitochondria and granular endoplasmic reticulum of the Schwann cell are involved in the production and storage of proteins for the plasma membrane of this cell, and with the hypothesis that these organelles are involved in the production and storage of protein metabolites which are subsequently transferred to the related axons.     

Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.     

Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

FDA and NIST collaboration on standards development activities supporting innovation and translation of regenerative medicine products

The development of standards for the field of regenerative medicine has been noted as a high priority by several road-mapping activities. Additionally, the U.S. Congress recognizes the importance of standards in the 21st Century Cure Act. Standards will help to accelerate and streamline cell and gene therapy product development, ensure the quality and consistency of processes and products, and facilitate their regulatory approval. Although there is general agreement for the need of additional standards for regenerative medicine products, a shared understanding of standards is required for real progress toward the development of standards to advance regenerative medicine. Here, we describe the roles of standards in regenerative medicine as well as the process for standards development and the interactions of different entities in the standards development process. Highlighted are recent coordinated efforts between the U.S. Food and Drug Administration and the National Institute of Standards and Technology to facilitate standards development and foster science that underpins standards development.     

Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time‐dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 µM . Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time‐dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration‐dependent, which correlates to the concentration‐dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration‐dependent killing is driven by the extent of the immediate permeabilizing action of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Secretome Analysis of Hypoxia‐Induced 3T3‐L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte‐secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3‐L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin‐1 and ‐2, and matrix metalloproteinase‐11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3‐L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.     

Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions

The isolation of chitosan from a fungal source offers the potential of a product with controlled physicochemical properties not obtainable by the commercial chemical conversion of crustacean chitin. A variety of culture and processing protocols using Mucor rouxii were studied for their effects on biomass yield and chitosan molecular weight. Weight-averaged molecular weight determined by gel permeation chromotography ranged from 2.0 x 10(5) to approximately 1.4 x 10(6) daltons. The chitosan yield ranged from 5% to 10% of total biomass dry weight and from 30% to 40% of the cell wall. Of the culture parameters studied, length of incubation and medium composition effected biomass production and molecular weight. Modification of the processing protocol, including the type and strength of acid, and cell wall disruption in acid prior to refluxing were used to optimize the efficiency of chitosan extraction.The degree of deacetylation of fungal and commercial chitosans was compared using infrared spectrometry, titration, and first derivative of UV absorbance spectrometry. The chitosan obtained directly from the fungal cell wall had a higher degree of deacetylation than commercial chitosan from the chemical conversion process.     

Immobilization and orientation‐dependent activity of a naturally occurring antimicrobial peptide

A naturally occurring antimicrobial peptide, SMAP‐29, was synthesized with an n‐terminal or c‐terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site‐directed immobilization to superparamagnetic beads. Immobilized SMAP orientation‐dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time‐dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub‐lethal effects. Immobilized SMAP activity was equivalent or reduced compared with soluble SMAP_c and c_SMAP regardless of immobilization orientation, with only one exception. A comparison of immobilized SMAP_c and c_SMAP activity revealed a bacteria‐specific potency dependent on immobilization orientation, which was contrary to that seen in solution, wherein SMAP_c was more potent against all bacteria than c_SMAP. Sub‐MBC kinetic studies displayed the influence of peptide exposure to the cells with multiple bacteria exhibiting increased susceptibility and efficacy at lower concentrations upon extended exposure (i.e. MBC enhancement). For instances in which complete killing was not achieved, two predominant effects were evident: retardation of growth rate and an increased lag phase. Both effects, seen independently and concomitantly, indicate some degree of induced cellular damage that can serve as a predictor toward eventual cell death. SMAP_c immobilized on glass through standard silanization chemistry was also investigated to ascertain the influence of substrate on activity against select bacteria. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.