Biocalorimetry- supported analysis of fermentation processes

The close relation between metabolic activity and heat release means that calorimetry can be successfully applied for on-line monitoring of biological processes. Since the use of available calorimeters in biotechnology is difficult because of technical limitations, a new sensitive heat-flux calorimeter working as a laboratory fermenter was developed and tested for different aerobic and anaerobic fermentations with Saccharomyces cerevisiae and Zymommonas mobilis. The aim of the experiments was to demonstrate the abilities of the method for biotechnological purposes. Fermentations as well as the corresponding heat, substrate and product analyses were reproducible. During experiments the heat signal was used as a sensitive and fast indicator for the response of the organisms to changing conditions. One topic was the monitoring of diauxic growth phenomena during batch fermentations, which may affect process productivity. S. cerevisiae was used as the test organism and a protease-excreting Bacillus licheniformis strain as an industrial production system. Other experiments focused on heat measurements in continuous culture under substrate-limiting conditions in order to analyse bacterial nutrient requirements. Again, Z. mobilis was used as the test organism. Ammonium, phosphate, magnesium, biotin and panthothenate, as important substrate compounds, were varied. The results indicate that these nutrients are required in lower amounts for growth than formerly suggested. Thus, a combination of heat measurements and other methods may rapidly improve our knowledge of nutrient requirements even for a well-known microorganism like Z. mobilis. *** DIRECT SUPPORT *** AG903062 00004     

Further studies on satellite nucleoli in rat and mouse hepatocytes

To provide more information on satellite nucleoli, these nuclear structures were studied by means of cytochemical and immunofluorescence procedures in rat and mouse hepatocytes without and following experimental inhibition of the RNA synthesis. The immuno-staining specific for nucleoli or B23 as well as C23 proteins demonstrated that satellite nucleoli and characteristic nucleoli exhibit the same fluorescence. The number of satellite nucleoli decreased after inhibition of nucleolar RNA synthesis in a similar way to the number of silver-stained granules (SSGs) of characteristic nucleoli. Inhibition of RNA synthesis also reduced the number of hepatocytes containing satellite nucleoli. Thus, satellite nucleoli seem to be real nucleoli from single NORs which did not fuse in the formation of a characteristic nucleolus.     

The primary nucleotide sequence of U4 RNA

U4 RNA is one of the "capped" nuclear snRNAs recently found to be precipitable by anti-Sm antibodies as ribonucleoprotein particles. U4 RNA, along with other snRNAs, has been implicated in hnRNA processing, mRNA transport, or both (Lerner, M. R., Boyle, J., Mount, S., Wolin, S., and Steitz, J. A. (1980) Nature 283, 220-224). Since the proteins bound to different snRNAs appear to be the same, the functions of different snRNPs might be dependent on the RNA components. To help understand the function of U4 RNP, the nucleotide sequence of U4 RNA was determined. The sequence is (formula see text) In addition to the modified nucleotides in the "cap," U4 RNA contains Am at position 63 and m6A at position 98. It also exhibited A-C microheterogeneity at position 97.     

Differences in chromatin proteins of resting and growing human lymphocytes.

Two-dimensional polyacrylamide gel electrophoresis comparisons were made for the non-histone “Chromatin fraction II” proteins of normal, phytohemagglutinin-stimulated and acute leukemic lymphocytes. The “Chromatin fraction II” proteins were extracted from the nuclear residue fraction after initial treatment with (a) 0.075 M NaCl containing 0.025 M EDTA, pH 8; (b) 0.01 M Tris-HCl, pH 8; and (c) 0.4 N H₂SO₄. Most of the proteins found earlier in the “Chromatin fraction II” of rodent liver and hepatomas were also found in the human cells. Some changes such as the decrease in amount of protein BA of normal rodent cells were found in the comparison of normal and stimulated human cells. By comparison with normal lymphocytes, the phytohemagglutinin-treated cells had decreased spot densities and sizes for proteins BA and Bv and an increase in densities and sizes of proteins CB, C25, CS and CT. In the acute lymphocytic leukemic cells there was a decrease in spots A24, BA, Bv, CD and CD′ by comparison with the normal lymphocytes. Protein CG′ which was found earlier in the hepatomas was found in acute lymphocytic leukemic cells but not in the control or phytohemagglutinin-treated cells. These studies show that there is a loss in specific Chromatin proteins BA and Bv from the Chromatin of rapidly turning over cells. Concomitantly, increases are observed for the amounts of protein spots CB, C25, CS and CT in the actively growing cell samples.     

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Measurement of cell proliferation by heavy water labeling

DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.