Immobilized-enzyme continuous-flow reactor incorporating continuous electrochemical regeneration of NAD

Electrochemical regeneration of the cofactor nicotinamide adenine dinucleotide (NAD) from its reduced form (NADH) has been coupled with the alcoholdehydrogenation reaction which consumes NAD and produces NADU using alcohol dehydrogenase bound to alumina. Alcohol (reactant) is added directly to the system while aldehyde (product) leaves the system through an ultrafiltration membrane which prevents loss of the cofactor. This system provides a continuous-flow process for carrying out a cofactor-requiring enzymatic reaction with no net loss or consumption of enzyme or cofactor and without the use of reagents for regenerating the cofactor. Although the process shown here is not economically practical, it may be a harbinger of useful and technically feasible chemical reaction systems based on immobilized enzymes requiring cofactors.     

Cultured endothelial cells derived from the human iliac arteries

Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins.     

Cultured endothelial cells derived from the human iliac arteries

Summary Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins. The above work was supported by Grant CA28540 from the National Institutes of Health and by a grant from The Council for Tobacco Research, USA.     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

Trauma and trephination in a Peruvian mummy

A multidisciplinary team participated in the examination of a colonial era Peruvian mummy. This young adult male had suffered multiple cranial and facial wounds and fractures and he died soon after trephination. Other pathologic findings were limited to the lungs, which showed anthracosilicosis and possible fibrosis. The observed pathology is linked to the two major health hazards of the early colonial era, physical trauma and silver mining. There was no evidence of any benign or malignant tumor, consistent with previous studies of ancient human remains. Such comparisons of ancient and modern mortality are intended to provide a historial perspective on the evolution and etiology of human disease, eventually on a population basis.     

Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium-alginate beads: I. Batch and fed-batch fermentations

In batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized-cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell-free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.     

The effect of neonatal treatment of rats with nerve growth factor on the blood pressure and structure of the mesenteric arteries.

Newborn male Wistar rats were treated with nerve growth factor daily by subcutaneous injection for 2 weeks, and control rats were treated with either cytochrome c or buffered saline. Average body weight of the treated animals was lower than that of the controls during the 2 weeks of treatment, but became similar to that of the controls thereafter. Tissue levels of norepinephrine were elevated in the brain, adrenal glands, mesenteric arteries, and vas deferens of the treated animals immediately after the treatment, but became similar in the three groups 2 weeks after the termination of the treatment. Blood pressure and heart rate were measured beginning at 4 weeks of age until 28 weeks, when the rats were sacrificed and the mesenteric arteries sampled for morphometric measurements of vessel wall dimensions. Pretreatment with nerve growth factor did not affect blood pressure, nor heart rate. Structural alteration of the three types of mesenteric arteries was also absent in the treated animals. We conclude that even though neonatal treatment of normal Wistar rats with nerve growth factor for 2 weeks induced an elevation of the norepinephrine levels in several tissues at the end of the treatment period, it was not sufficient to produce hypertension and structural alterations in the blood vessels.     

The evolutionary impacts of conservation actions

Conservation management for environmental sustainability is now ubiquitous. The ecological effects of these actions are well-intentioned and well-known. Although conservation biologists and managers increasingly incorporate evolutionary considerations into management plans, the evolutionary consequences of management strategies have remained relatively unexplored and unconsidered. But what are the evolutionary consequences? Here, we advocate a new research agenda focused on identifying, predicting, and countering the evolutionary consequences of conservation management. We showcase the examples of park creation and invasive species management, and speculate further on five other major methods of management. Park creation may cause selection for altered dispersal and behavior that utilizes human foods and structures. Management of invasive species may favor the evolution of resistance to or tolerance of control methods. In these and other cases, evolution may cause deviations from the predicted consequences of management strategies optimized without considering evolution, particularly when management results in or coincides with major environmental change, if population size change strongly, or if life histories are short enough to allow more rapid evolution. We call for research focused on: (1) experimental predictions and tests of evolution under particular management strategies, (2) widespread monitoring of managed populations and communities, and (3) meta-analysis and theoretical study aimed at simplifying the process of evolutionary prediction, particularly at systematizing a means of identifying traits likely to evolve due to likely existing genetic variance or high mutation rates. Ultimately, conservation biologists should incorporate evolutionary prediction into management planning to prevent the evolutionary domestication of the species that they are trying to protect.     

Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent

1. Download high-res image (226KB)
2. Download full-size image

     

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.