Monitoring the rate of oxygen consumption in plant cell suspensions

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in the rate of chitin-plus-chitosan synthesis accompany morphogenesis of Mucor racemosus.

The in vivo differential rates of chitin-plus-chitosan biosynthesis in Mucor racemosus were determined under a variety of conditions, leading to yeast cell or mycelial morphology. Chitin-chitosan was determined as hot NaOH-insoluble radioactivity derived from N-acetyl-D-[1-3H]glucosamine in the medium. Control experiments demonstrated that the labeled material possessed the properties of chitin-plus-chitosan. Our results indicate that Mucor yeasts have a relatively low differential rate of chitin-plus-chitosan synthesis and that mycelial cells have a threefold-elevated differential rate. Treatment of aerobic cells with exogenous N6, O2-dibutyryl cyclic adenosine 3',5'-monophosphate, an agent which induces yeast cell morphology, also results in a lowered rate of chitin-plus-chitosan synthesis. Control experiments eliminated the possibility that the observed rate changes were due to changes in endogenous pool size, uptake of exogenous N-acetyl-p-[1-3H]glucosamine, or alterations in growth rate. Therefore, the changes are seemingly linked to morphogenesis. These results strengthen the idea that cyclic adenosine 3',5'-monophosphate plays an important role in dimorphism in Mucor. In addition, pulse-chase experiments suggest that considerable modification of newly synthesized chitin plus chitosan in both yeast cells and mycelia occurs in vivo.     

Preparation of Alexa Fluor 350-conjugated nonradioactive or 3H-labeled GM1 ganglioside derivatives with different ceramides

Alexa Fluor 350 hydrazide (AF) was coupled to the aldehyde group at C-6 of terminal galactose of oxidized GM1 gangliosides containing different fatty acid residues (GM1s). The AF-GM1 hydrazones obtained were reduced with NaBH₄ or [³H]NaBH₄ and purified by high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Final yields of AF-GM1s exceeded 30%, purity was better than 97%, and radiochemical purity of ³H-labeled AF-GM1s was more than 94.5%. Structures of AF-GM1s were confirmed by electrospray ionization-mass spectrometry (ESI-MS). When added to HL-60 cell culture media, more than 81.6 or 78.9% of the AF-[³H]GM1s were taken up by cells in a bovine serum albumin- or trypsin-resistant manner, respectively. Approximately 70% of the AF-[³H]GM1s were recovered in HL-60 total plasma membrane fraction.     

Effects of virus concentration and ultraviolet irradiation on the activity of corn earworm and beet armyworm (Lepidoptera: Noctuidae) nucleopolyhedroviruses

Laboratory studies were initiated to determine the relationship between virus concentration and radiation-caused inactivation of NPVs from Helicoverpa zea (HzSNPV) and Spodoptera exigua (SeMNPV). In the laboratory, a UV-B/UV-A system was used for inactivation studies. For both viruses inactivation was dependent upon both length of UV exposure and virus concentration. At all virus concentrations HzSNPV was more sensitive to UV than SeMNPV. In the field HzSNPV was used and virus persistence was significantly affected by virus concentration (i.e., inactivation was inversely related to virus concentration).     

Rigidification of a flexible protease inhibitor variant upon binding to trypsin

The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.     

Determination of antithyroid antibodies by an immunoenzymatic method; an adaptation for expressing results in international units]

Solid phase immunoenzymatic methods (ELISA) have been developed for the determination of antithyroglbulin (ATG), antimicrosomal (AMc) and antimembrane (ATMA) antibodies in blood serum. The results have been expressed in international units (IU). The level of nonspecific reaction was determined on the basis of 30 samples of blood serum obtained from healthy donors. The double standard deviation values amounted to 8 IU for antithyroglobulin antibodies, 17 IU for antimicrosomal antibodies and 53 IU for antimembrane antibodies at the serum dilution of 1:100. The values of double standard deviation obtained for the healthy donors correspond to the borderline between the positive serum samples and those containing no autoantibodies. The level of autoantibodies in patients with autoimmune diseases of the thyroid varied considerably ranging from complete absence to several thousand units per milliliter in single cases. Antithyroglobulin antibodies were determined simultaneously by using the described method and the commercial kit (Walker, Cambridge) and the results obtained by the two methods were compared. A linear correlation with the correlation coefficient r = 0.93, p < 0.001 was obtained. A good but nonlinear correlation was demonstrated with the methods expressing the results in titre values.     

Sequence and structural characterization of great salt lake bacteriophage CW02, a member of the T7-like supergroup

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.     

The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [³H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C₁₄, C₁₈ or C_18:1 fatty acids (Fas)     

Are there Characteristics of Infectious Diseases that Raise Special Ethical Issues?1

This paper examines the characteristics of infectious diseases that raise special medical and social ethical issues, and explores ways of integrating both current bioethical and classical public health ethics concerns. Many of the ethical issues raised by infectious diseases are related to these diseases' powerful ability to engender fear in individuals and panic in populations. We address the association of some infectious diseases with high morbidity and mortality rates, the sense that infectious diseases are caused by invasion or attack on humans by foreign micro-organisms, the acute onset and rapid course of many infectious diseases, and, in particular, the communicability of infectious diseases. The individual fear and community panic associated with infectious diseases often leads to rapid, emotionally driven decision making about public health policies needed to protect the community that may be in conflict with current bioethical principles regarding the care of individual patients. The discussion includes recent examples where dialogue between public health practitioners and medical-ethicists has helped resolve ethical issues that require us to consider the infected patient as both a victim with individual needs and rights and as a potential vector of disease that is of concern to the community.