Development of the Particle Inflow Gun

A simple and inexpensive particle acceleration apparatus was designed for direct delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles directly in a helium steam. High levels of transient expression of theβ-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of maize and soybean, and leaf tissue of cowpea. Stable transformation of soybean and maize has also been obtained using this bombardment apparatus.     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

High concentration active enzyme centrifugation: analysis of active polymeric forms at up to 10 000-fold higher concentrations than with conventional methods.

This paper describes the theoretical basis, experimental technique, and experimental evaluation of a new method of analysis called "high concentration active enzyme centrifugation". It extends by up to four orders of magnitude the upper concentration limits at which the technique of "active enzyme centrifugation" can be used for analysis of enzyme structure. This new theory is largely based on certain properties of Gaussian curves which we have described in previous publications [Wei, G.J., & Deal, W.C., Jr. (1976) Anal. Biochem. 75, 113-121; Anal. Biochem. (1978) 87, 433-446]. One of the most important aspects of this development is that it extends the concentration range upward so that experiments can be performed on enzymes in the active polymeric forms corresponding to their in vivo states. Furthermore, this expansion includes the range in which most enzymes go through all their association-dissociation transitions from one polymeric form to another. Hence, the method can be used to define the various concentration-dependent transitions and also to ascertain which of the various polymeric forms of an enzyme are active, under various conditions. This method also retains the many favorable characteristics inherent in the active enzyme centrifugation technique. In studies with lactate dehydrogenase, the results from this method of band sedimentation were identical within experimental error (about 1.5%) with results from conventional boundary sedimentation velocity studies.     

Indoleacetic acid oxidase activity in main stem homogenates of flax genotrophs and genotypes

The IAA-oxidase and peroxidase capabilities along the length of the main stem tissues of two flax genotrophs L and S and two flax genotypes R and M were examined in vitro. Stem gradients for peroxidase activity increased basipetally in all plant types, as did IAA-oxidase activity gradients at non-rate-limiting concentrations of Mn²⁺. Correlations between peroxidase activity and non-rate-limited IAA-oxidase activity supported the contention of dual activities on the same molecule. At rate-limiting concentrations of Mn²⁺, IAA-oxidase activity did not correlate with peroxidase activity. Plant type differences were detected in rate-limited IAA-oxidase activity. This activity was higher in the stem region immediately above the cotyledons (axillary buds) of the more branched types, L and R, than in the sparsely branched types, S and M.     

Synthesis, conjugation, and radiolabeling of a novel bifunctional chelating agent for (225)Ac radioimmunotherapy applications

225Ac (t(1/2) = 10 days) is an alternative alpha-emitter that has been proposed for radioimmunotherapy (RIT) due to its many favorable properties, such as half-life and mode of decay. The factor limiting use of (225)Ac in RIT is the lack of an acceptably stable chelate for in vivo applications. Herein is described the first reported bifunctional chelate for (225)Ac that has been evaluated for stability for in vivo applications. The detailed synthesis of a bifunctional chelating agent 2-(4-isothiocyanatobenzyl)-1,4,7,10,13, 16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (HEHA-NCS) is reported. This ligand was conjugated to three monoclonal antibodies, CC49, T101, and BL-3 with chelate-to-protein ratios between 1.4 and 2. The three conjugates were radiolabeled with (225)Ac, and serum stability study of the [(225)Ac]BL-3-HEHA conjugate was performed.     

Coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization

Despite their high frequency of RNA recombination, the plus-strand coronaviruses have a characteristic, strictly conserved genome organization with the essential genes occurring in the order 5'-polymerase (pol)-S-E-M-N-3'. We have investigated the significance of this remarkable conservation by rearrangement of the murine coronavirus genome through targeted recombination. Thus, viruses were prepared with the following gene order: 5'-pol-S-M-E-N-3', 5'-pol-S-N-E-M-3', 5'-pol-M-S-E-N-3', and 5'-pol-E-M-S-N-3'. All of these viruses were surprisingly viable, and most viruses replicated in cell culture with growth characteristics similar to those of the parental virus. The recombinant virus with the gene order 5'-pol-E-M-S-N-3' was also tested for the ability to replicate in the natural host, the mouse. The results indicate that the canonical coronavirus genome organization is not essential for replication in vitro and in vivo. Deliberate rearrangement of the viral genes may be useful in the generation of attenuated coronaviruses, which due to their reduced risk of generating viable viruses by recombination with circulating field viruses, would make safer vaccines.     

Nasal and tracheal responses to chemical and somatic afferent stimulation in anesthetized cats

Respiratory chemical and reflex interventions have been shown to affect nasal resistance or tracheal tone, respectively. In the present study, nasal caliber (assessed from pressure at a constant flow) and tracheal tone (assessed from pressure in a fluid-filled balloon within an isolated tracheal segment) were monitored simultaneously in anesthetized, paralyzed, artificially ventilated (inspired O2 fraction = 100%) cats. We examined the effect of CO2 inhalation and sciatic nerve stimulation as well as the application of nicotine (6 X 10(-4) mol/l) or lidocaine (2% solution) to the intermediate area of the ventral medullary surface (VMS). CO2 and VMS nicotine resulted in a significant increase in tracheal pressure [147 +/- 73 and 91 +/- 86% (SD), respectively]; and a significant reduction in nasal pressure (-35 +/- 10 and -20 +/- 13%, respectively). In contrast, sciatic nerve stimulation resulted in a significant fall in both tracheal (-50 +/- 36%) and nasal pressure (-21 +/- 13%). Application of 2 or 4% lidocaine to the VMS reduced tracheal pressure but did not significantly affect nasal pressure. After VMS lidocaine, nasal and tracheal responses to CO2, sciatic nerve stimulation, or VMS nicotine, when present, were negligible. These results suggest a role for the VMS in the regulation and coordination of nasal and tracheal caliber responses.