MORPHOLOGY OF ISOLATED RABBIT HEART MUSCLE MITOCHONDRIA AND THE OXIDATION OF EXTRAMITOCHONDRIAL REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

The morphology of rabbit heart muscle mitochondria isolated in several media has been compared by electron microscopy. The internal structure of isolated mitochondria differs from that of in situ mitochondria, with the type and degree of alteration depending on the isolation medium. Examination of the isolated mitochondria after incubation revealed that additional morphological changes occurred during incubation, but these changes were less pronounced when the incubation was conducted in a complete medium containing substrate. The isolated mitochondria have been shown to be capable of catalyzing a slow aerobic oxidation of extramitochondrial reduced diphosphopyridine nucleotide. The rate of DPNH oxidation observed is sufficient to account for the ability of the mitochondria to oxidize lactate in the presence of catalytic amounts of DPNH. The suspensions used were essentially free of mitochondrial fragments, which are known to oxidize DPNH. Possible relationships of these findings to metabolism in situ are discussed. The results indicate the desirability of correlating biochemical activities with the morphology of isolated mitochondria.     

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.     

Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies.

In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC.     

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.     

Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.

Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

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.