Immobilized enzymes: Pectin esterase covalently coupled to dorous glass particles

Pectin esterase (E.C. 3.1.1.11) was covalently immobilized to porous glass particles by reaction of the native protein with pendant, benzoyl azide groups of the carrier. Enzyme loading on the carrier was 0.5 unit per ml as measured by pH stat, assay. Decreasing the size of the immobilized enzyme particles by grinding produced a 12-fold increase in activity suggesting severe internal mass transport restrictions on turnover kinetics, Gross fractionation of the citrus pectin substrate into high and low molecular weight categories and their subsequent use in kinetic characterization shows no effect of molecular weight upon the kinetic behavior of the native enzyme. In contrast the immobilized enzyme displayed a 5-fold increase in the apparent. K_m for the high molecular weight fraction relative to that of the low molecular weight fraction. A striking difference exists in the low pH profile of immobilized pectin esterase relative to the native enzyme. Carrier matrix interactions with the polyelectrolyte substrate are invoked to explain this difference. The thermal stability of the immobilized enzyme is relatively low and displays a half-life of approximately 2 weeks at 25°C.     

A stereologic electron microscope study of “tubular myelin figures” in alveolar fluids of rat lungs

Summary The three-dimensional structure of a composite material found in alveolar exudate of oxygen poisoned lungs but also present in normal lungs is stereologically analysed. It is composed of tubules of 450 Å diameter which are tightly packed in a quadratic lattice. The wall of the tub vile is formed by four-winged osmiophilic filaments which are located in the corners of the quadratic lattice; their interior is made up of a hydrophilic substance which contains either a tubule or a filament of moderate electron density. The osmiophilic substance of the walls is continuous with associated myelin figures which can be resolved into lamellae with a periodicity of 42 Å and can thus be considered to be water crystals of phospholipids. The nature of the content of the tubules, which presumably exerts the formative force on the phospholipid lamellae to form tubules, remains undetermined.Dedicated to Prof. W. Bargmann in honor of his 60th birthday.The research reported here has been sponsored by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (Nr. 2569); by the Stiftung für wissenschaftliche Forschung an der Universität Zürich; by the National Institutes of Health, USPHS, through grant RF-57; and the 6570th Aerospace Medical Research Laboratories under contract AF 61(052)-784 through the European Office of Aerospace Research (OAR), United States Air Force.     

A MORPHOMETRIC STUDY ON THE THICKNESS OF THE PULMONARY AIR-BLOOD BARRIER

A reliable knowledge of the thickness of the alveolo-capillary "membrane" or air-blood barrier is of physiologic interest since it is intimately related to a quantitative estimation of such functional events as gas diffusion or tissue metabolism in the lung. The characteristic thickness of the air-blood barrier with respect to gas diffusion is its harmonic mean thickness, while the arithmetic mean thickness is related to the mass of tissue building the barrier and consuming oxygen in the lung. Two morphometric methods are proposed by which these two dimensions can be estimated from random measurements in the electron microscope in a reliable, simple, and efficient manner. By applying these methods to three rat lungs the arithmetic mean thickness of the barrier was found to measure 1.25 µ, the harmonic mean thickness, 0.57 µ. On the basis of these measurements a geometric model of the barrier in the form of a corrugated membrane was derived. Its dimensions showed close similarity to those of the natural barrier. This analysis suggested furthermore that the gas conductance of the barrier is nearly optimal if one considers the mass of tissue and the minimal barrier thickness as fixed properties which are determined by other functional requirements on the alveolo-capillary membrane.     

Bacterial Cellulose as a Substrate for Microbial Cell Culture

Bacterial cellulose (BC) has a range of structural and physicochemical properties that make it a particularly useful material for the culture of bacteria. We studied the growth of 14 genera of bacteria on BC substrates produced by Acetobacter xylinum and compared the results to growth on the commercially available biopolymers agar, gellan, and xanthan. We demonstrate that BC produces rates of bacterial cell growth that typically exceed those on the commercial biopolymers and yields cultures with higher titers of cells at stationary phase. The morphology of the cells did not change during growth on BC. The rates of nutrient diffusion in BC being higher than those in other biopolymers is likely a primary factor that leads to higher growth rates. Collectively, our results suggest that the use of BC may open new avenues in microbiology by facilitating bacterial cell culture and isolation.     

Profiling of bacterial adhesin--host receptor recognition by soluble immunoglobulin superfamily domains

Several gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa(CEA) proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa(CEA)-expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen-receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.     

Melatonin or a melatonin agonist corrects age-related changes in circadian response to environmental stimulus

The effects of a melatonin agonist, S-20098, included in the diet were tested on a specific effect of aging in hamsters: the marked decline in the phase shifting effects of a 6-h pulse of darkness on a background of constant light. In contrast to young hamsters, old hamsters fed with the control diet showed little or no phase shifts in response to a dark pulse presented in the middle of their inactive or active period. Old hamsters fed with S-20098 showed phase shifts that were ~70% of the ones in young animals and significantly greater than those in old controls. The phase advancing response to a dark pulse presented during the inactive period was dose dependent and reversed after S-20098 discontinuation. Melatonin included in the diet showed comparable restorative effects on the phase shifting response to a dark pulse in old hamsters. Replacement therapy with melatonin or melatonin-related compounds could prove useful in treating, preventing, or delaying disturbances of circadian rhythmicity and/or sleep in older people.