Use of comparative reasoning tools for evaluation of the effect of sterilization conditions on fermentations to produce acidic fibroblast growth factor

The effects of various medium sterilization conditions on fermentations of a recombinant, acidic fibroblast growth factor (aFGF) producing Escherichia coli have been studied. Changes in the medium resulting from sterilization were monitored by pH and absorption spectra. This simple experiment provided excellent data for the demonstration of the usefulness of comparative reasoning tools in order to evaluate the effect of sterilization on fermentation performance. The time profiles of the main parameters (e.g., carbon dioxide evolution rate, dissolved oxygen, pH, and aFGF productivity) were simplified into piecewise contiguous linear segments, each of which was sequentially numbered. The length, position, and slope of each tine were then characterized. Application of the comparative reasoning tools confirmed that separate sterilization of the glucose was necessary for the success of the process, despite adding to the cost and complexity. The comparative data analysis also showed that scaleup with longer sterilization holding and cooling times would not be detrimental to aFGF production. (c) 1995 John Wiley & Sons, Inc.     

Factors involved in capillary growth in the heart

Growth of capillaries in the heart occurs under physiological circumstances during endurance exercise training, exposure to high altitude and/or cold, and changes in cardiac metabolism or heart rate elicited by modification of thyroid hormone levels. Capillary growth in all these conditions can be linked with increased coronary blood flow, decreased heart rate, or both. This paper brings evidence that, although increased blood flow due to long-term administration of coronary vasodilators results in capillary growth, a long-term decrease in heart rate induced by electrical bradycardial pacing in rabbits and pigs, or by chronic administration of a bradycardic drug, alinidine, in rats, stimulates capillary growth with little or no change in coronary blood flow. Decreased heart rate results in increased capillary wall tension, increased end-diastolic volume and increased force of contraction, and thus stretch of the capillary wall. This could lead to release of various growth factors possibly stored in the capillary basement membrane. Correlation was found between capillary density (CD) and the levels of low molecular endothelial cell stimulating angiogenic factor (ESAF) both in rabbit and pig hearts with CD increased by pacing. There was no relation between expression of mRNA for basic fibroblast growth factor and CD in sham-operated and paced rabbit hearts. In contrast, mRNA for TGFß was increased in paced hearts, and the possible role of this factor in the regulation of capillary growth induced by bradycardia is discussed.     

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme

We enzymatically deglycosylated pig lung angiotensin I-convertingenzyme (ACE) to study the involvement of its glycanic chainsin its physicochemical and catalytic properties. The effectsof endoglycosidases F₂ and H, and of N-glycanase were assessedby ACE mobility in SDS-PAGE. N-Glycanase only was completelyeffective with or without previous denaturation, leading toa shift in ACE M_r from 172 to 135 kDa; endoglycosidase F₂ producedthe same shift but only without previous denaturation. DeglycosylatedACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine,and an identical Stokes radius as measured by size-exclusionhigh performance liquid chromatography. Neuraminidase had noeffect on ACE Stokes radius but slightly decreased its kcatwhich could be related to variations in ionization of the activesite. The isoelectric point of ACE, as, determined by isoelectricfocusing, increased from 4.5–4.8 to 5.0–5.3 aftereither endoglycosidase F₂ or neuraminidase digestion, but stillwith microheterogeneities which thus did not seem to be relatedto ACE glycans. Deglycosylated ACE did not bind onto agaroselectinsin contrast to native ACE which bound strongly to concanavalinA showing interactions involving oligomannosidic or biantennaryand sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin,an inhibitor of N-glycosylation, did not modify ACE secretionby endothelial cells. Thus, ACE glycans have no drastic effectson structural and biological properties of the protein, butthey may have a functional role on intracellular targeting ofboth secreted and membrane-bound ACE isoforms, also for theprotection of the soluble plasma form against hepatic lectinsand the maintenance of its hydrosolubility. converting enzyme (peptidyldipeptidase EC.3.4.15.1) endothelium glycosidases lectins     

Nuclear envelope assembly after mitosis

In higher eukaryotes, the entire nucleus disassembles during prometaphase of the cell cycle and later reassembles around daughter chromosomes. Remarkably, the complex events that occur to create a functional nucleus in vivo can be duplicated in vitro by using cell-free extracts. Current experiments are aimed at understanding the molecular mechanisms of assembly and disassembly of the nuclear pore complexes and nuclear membranes, and the functional roles of four identified inner membrane proteins, two of which bind to both chromatin and the nuclear lamina.     

Microspectrophotometric studies of Romanowsky stained blood cells. IV. Maturation of myeloid and erythroid cell lines in bone marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the "true" classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.     

A genetic analysis of natural resistance to nonsyngeneic cells: The role of H-2

The genetic control of natural resistance in vivo to four natural killer (NK) cell-resistant H-2 homozygous lymphoid tumor cell lines was investigated by following the survival and organ distribution of cells prelabeled with radioactive iododeoxyuridine. Backcross mice derived from DBA/2J and CBA/J parents were injected with H-2 ^dtumor cells and tumor cell elimination was lowest in H-2 ^dhomozygotes. Natural killer cell activity was also reduced in mice with the H-2 ^dhaplotype, but no direct correlation between NK cell levels against YAC-1 or SL2-5 lymphoma cells and natural resistance in vivo was demonstrable. Analysis of 23 BXD recombinant inbred strains indicated that natural resistance to H-2 ^dtumors was restricted to H-2 ^bstrains. There was no direct association of NK cell activity with H-2 type in the BXD strains and NK cell levels did not correlate with tumor survival in vivo. By comparing natural resistance to H-2 ^dand H-2 ^btumors in DBA/2, C57BL/6, B6D2F₁, and B10.D2 mice we found that H-2 nonidentity between the tumor and the host, rather than the host H-2 haplotype, determined whether natural resistance occurred. Again, NK cell activity against YAC-1 cells was not predictive of tumor survival in these strains. These results provide genetic evidence that NK cells alone cannot account for natural resistance to H-2 nonidentical cells of hemopoietic origin.     

A modified direct phosphate assay for studying ATPases

A simple, rapid assay for purified ATPases is presented, based upon the formation of phosphomolybdate and its extraction into butyl acetate. The inclusion of imidazole makes the assay more sensitive and reproducible apparently because of the formation of an imidazole-phosphomolybdate complex. Protein (100 micrograms), Hepes buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (0.1 M) and nucleotides (1 mM) were all shown to cause interference. The interference by nucleotides could be counteracted by using more molybdate. Butyl acetate was shown to extract virtually all of the phosphomolybdate almost instantaneously upon vortex mixing.     

Gecko phonotaxis to cricket calling song: A case of satellite predation

Insectivorous Mediterranean house geckos, Hemidactylus tursicus, are found in the field close to the burrows of calling male decorated crickets, Gryllodes supplicans. In playback experiments where adult geckos were presented with calls of these crickets or calls of frogs as controls, geckos exhibited positive phonotaxis to broadcast cricket calling song. This indicates that geckos orient to and approach male cricket calls, even though the crickets call from burrows where they are protected from the geckos. However, this behaviour enables the geckos to intercept and consume female crickets that also respond phonotactically to the cricket calls. Thus geckos act as ‘satellite predators’, a situation which may impose sex-biased mortality on female crickets.