Production of 2-Ketogluconic Acid by Serratia marcescens

Production of 2-ketogluconic acid from glucose by fermentation with Serratia marcescens NRRL B-486 was studied in 20-liter stainless-steel fermentors. Conditions for 2-ketogluconic acid production included the following: glucose-salt medium, aeration rate of 0.75 volumes per volume per minute, agitation rate of 400 rev/min, temperature of 30 C, CaCO₃ to neutralize the acid formed, and a 5% (v/v) inoculum. Foaming was controlled with an antifoam agent added at intervals during the fermentation. When 120 g per liter of glucose were supplied, 95 to 100% yields of 2-ketogluconic acid were obtained in 16 hr. Larger amounts of glucose could be used in the fermentation provided that the carbohydrate was fed continuously. Continuous feeding of glucose to a total amount of 180 g per liter gave 95 to 100% yields of 2-ketogluconic acid in 24 hr; feeding glucose to a total amount of 240 g per liter gave 85 to 90% yields in 32 to 40 hr.     

Growth-related changes of oxygen consumption rates of tumor cells grown in vitro and in vivo

Growth-related changes of oxygen consumption rates of tumor cells, grown in vitro or in vivo, were investigated. For in vitro investigations, L929 and DS-carcinosarcoma cells were cultured in artificial media. For in vivo studies, DS-carcinosarcoma cells were implanted into the abdominal cavity of Sprague-Dawley rats (ascites tumor, containing malignant cells, leukocytes, lymphocytes, and macrophages). Oxygen uptake was measured photometrically. Parameters of the extracellular medium judged to possibly influence the respiratory activity of tumor cells were monitored at different growth stages (glucose, lactate, and amino acid levels, oxygen and carbon dioxide partial pressures, and pH values). The results obtained clearly show that the oxygen uptake of tumor cells grown in vitro decreased as quiescence developed. In contrast, the respiratory activity of in vivo DS-carcinosarcoma ascites cells increased as tumor growth reached plateau phase. The differences observed cannot be attributed solely to changes of the environmental conditions monitored. It is likely that an increased respiration rate of activated host cells might profoundly contribute to the elevation of the respiratory capacity of DS-carcinosarcoma ascites tumors grown in vivo. These data provide evidence that solid tumors in vivo can increase their O2 uptake at an enhanced O2 availability not only due to an enlarged tumor volume with adequate O2 supply but also due to an elevation of the respiratory activity of different cell populations within a tumor.     

Significant extension in northern Australia of the known geographic range of the Shield Shrimp Triops australiensis (Crustacea: Notostraca)

The program BIOCLIM predicts the total geographic distribution of species, based upon the biogeoclimatic characteristics common to the localities at which they are known to occur. Field studies in the Northern Territory have located the Shield Shrimp Triops australiensis at localities substantially north of its known and predicted geographic distribution.     

Lymphocytes protect against and are not required for reovirus-induced myocarditis.

Many studies suggest that host lymphocytes are damaging, rather than protective, in virally induced myocarditis. We have investigated the role of lymphocyte-based immunity in murine myocarditis by using a myocarditic reovirus (reovirus serotype 3 8B), nonmyocarditic reoviruses, adoptive transfer experiments, and mice with severe combined immunodeficiency (SCID mice). Prior to infection, passive transfer of monoclonal antibodies specific for 8B capsid proteins protected neonatal mice against 8B-induced myocarditis, indicating that humoral immunity can protect against myocarditis. Some monoclonal antibodies acted by blocking viral spread to and/or replication in the heart. Passive transfer of reovirus-immune, but not naive, spleen cells prior to infection protected neonatal mice from 8B-induced myocarditis. Depletion of either CD4 or CD8 T cells resulted in increased viral titer in the heart but did not abrogate immune cell-mediated protection against myocardial injury. This shows that both CD4 and CD8 T cells can act independently to protect myocardial tissue from reovirus infection. In addition, reovirus 8B caused extensive myocarditis in SCID mice. This confirms a prior report (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989) that T cells are not required for reovirus-induced myocarditis and demonstrates for the first time that B cells are not required for reovirus-induced myocarditis. We used SCID mice and a panel of reoviruses to assess (i) the relationship between growth in the heart and myocardial damage and (ii) the possibility that nonmyocarditic reoviruses exhibit a myocarditic phenotype in the absence of functional lymphocytes. Growth in the heart was not the sole determinant of myocarditic potential in SCID mice. Although 8B induced myocarditis in SCID mice, no or minimal myocarditis was found in SCID mice infected with four reovirus strains previously shown (B. Sherry and B. N. Fields, J. Virol. 63:4850-4856, 1989) to be nonmyocarditic or poorly myocarditic in normal neonatal mice. We conclude that (i) humoral immunity and cellular immunity are protective against, and not required for, reovirus-induced myocarditis and (ii) the potential to induce cardiac damage is a property of the virus independent of lymphocyte-based immunity.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460