Selection of a proteolytic enzyme to solubilize lean beef tissue

Many proteases are available for the hydrolysis of various protein substrates. The qualitative effect of most experimental variables on reaction progress is known, so it is possible to devise a rational procedure for selecting the best enzyme. Reaction time and enzyme concentration should be chosen in the region where they have little effect on reaction progress. Substrate concentration should be low to avoid possible product inhibition. Each enzyme should be tested at its optimum pH, and at a range of temperatures around (mainly below) the reported temperature optimum. Enzyme cost and other relevant factors should also be considered in the enzyme selection. Using this selection procedure Alcalase was chosen as the most appropriate enzyme for solubilizing lean beef tissue.     

Morphologic and pathologic findings in teeth and jaws from the middle bronze age (Pitten, Lower Austria)

The detailed examination of the masticating apparatus of 18 skulls from the Middle Bronze Age in Pitten (Lower Austria) revealed numerous pathological findings of the teeth. Most remarkable were frequency and extent of dental abrasions. In addition to it, indications on inflammatory processes in the marginal parodontium were obtained, combined with partly immense formations of concrements on the surfaces of the teeth. The incidence of dental caries was relatively low. Abnormal positions of the teeth and pathological processes concerning the development of dentition as well as the eruption of the wisdom teeth could be observed repeatedly. Conclusions on insufficiences of the oral hygiene in this time follow especially from the concrement findings and from the inflammatory reactions of the marginal parodontium.     

Clearance and fate of leukemia-inhibitory factor (LIF) after injection into mice

Leukemia-inhibitory factor (LIF) elicits effects on a broad range of cell types, including cells of the monocytic and megakaryocytic series, embryonal stem cells, hepatocytes, adipocytes, and osteoblasts. Native and recombinant LIF, injected intravenously into adult mice, had an initial half-life of 6-8 min and a more prolonged second clearance phase. Clearance of 125I-LIF from the circulation was paralleled by a rapid accumulation in the kidneys, liver, lungs, and spleen and a more gradual accumulation in the thyroid gland. Labeling of the renal glomerular tufts, parenchymal hepatocytes, splenic red pulp, alveolar pneumocytes, and thyroid follicular cells as well as of megakaryocytes and osteoblasts in the bone cavities, placental trophoblasts, and cells of the choroid plexus was demonstrable autoradiographically. The appearance of a large amount of nonprecipitable 125I in the urine suggested that the kidneys were the major route of LIF clearance from the body.     

Assessment of counting efficiencies for labeled protein and other macromolecules in filter disk assays

A several-fold greater counting efficiency is observed for protein labeled with [³H]leucine than for free [³H]leucine using a conventional filter disk assay. A similar, though less marked, effect is noted for ¹⁴C-labeled molecules. These results are comparable to those reported by others for counting efficiencies of labeled DNA and deoxynucleotides and illustrate the generality of this effect with regard to macromolecules and their low-molecular weight precursors. This phenomenon, presumably due to differences in the distribution of large and small molecules within filters, gives rise to errors in the quantitation of macromolecule synthesis if a counting efficiency identical to that of the precursor is assumed to apply. A convenient method for determining counting efficiencies of various molecules bound to filters is presented which eliminates this problem.     

Plasma immunoreactive melanotrophic hormones in patients on maintenance haemodialysis.

Circulating levels of melanotrophic horomones and ACTH were determined in patients treated by maintenance dialysis for chronic renal failure. Plasma melanotrophic hormone levels were greatly increased in all patients studied (125-1100 ng/1 as compared with 12-36 ng/1 in normal adults) and were correlated with the duration of treatment. Skin pigmentation, especially in exposed areas, was notably increased, particularly in those patients with the highest plasma melanotrophic concentrations. Plasma ACTH levels were normal or only slightly raised and circulating corticosteroid concentrations, as determined by a fluorimetric method, all lay within the physiological range. The dissociation between ACTH and melanotrophic hormone levels in these patients may have been the result of a slower metabolic clearance of the latter.     

Three-dimensional gas exchange pathways in pome fruit characterized by synchrotron x-ray computed tomography

Our understanding of the gas exchange mechanisms in plant organs critically depends on insights in the three-dimensional (3-D) structural arrangement of cells and voids. Using synchrotron radiation x-ray tomography, we obtained for the first time high-contrast 3-D absorption images of in vivo fruit tissues of high moisture content at 1.4-microm resolution and 3-D phase contrast images of cell assemblies at a resolution as low as 0.7 microm, enabling visualization of individual cell morphology, cell walls, and entire void networks that were previously unknown. Intercellular spaces were always clear of water. The apple (Malus domestica) cortex contains considerably larger parenchyma cells and voids than pear (Pyrus communis) parenchyma. Voids in apple often are larger than the surrounding cells and some cells are not connected to void spaces. The main voids in apple stretch hundreds of micrometers but are disconnected. Voids in pear cortex tissue are always smaller than parenchyma cells, but each cell is surrounded by a tight and continuous network of voids, except near brachyssclereid groups. Vascular and dermal tissues were also measured. The visualized network architecture was consistent over different picking dates and shelf life. The differences in void fraction (5.1% for pear cortex and 23.0% for apple cortex) and in gas network architecture helps explain the ability of tissues to facilitate or impede gas exchange. Structural changes and anisotropy of tissues may eventually lead to physiological disorders. A combined tomography and internal gas analysis during growth are needed to make progress on the understanding of void formation in fruit.     

Molecular basis for substrate recognition and septum cleavage by AtlA,the major N-acetylglucosaminidase of Enterococcus faecalis

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA–substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.     

New concepts for rapid yeast settling. II. pH switching with an inert powder

A new technique is outlined for the rapid settling of yeast cells in fermentation media. The technique involved the addition of dense, inert particles (nickel powder) to a yeast suspension (Saccharomyces cerevisiae) at pH 4.5 and a rapid change of pH to 8.0-9.0. When the pH was changed large flocs formed immediately and settled rapidly, leaving a clear supernatant. On returning the pH to 4.5 the flocs were destroyed. This technique gave larger flocs and higher settling rates than the constant pH method, and much lower nickel/yeast ratios were required. Good flocculation also occurred in a fermentation medium. The technique was used to recycle yeast cells to a semicontinuous ethanol fermentation. Application of the technique to this and similar systems is discussed. The factors affecting yeast/inert powder flocculation are also discussed and a model is proposed to explain the observed experimental behavior for flocculation with a rapid change in pH.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009