Effects of storage time and temperature on the infectivity and effectiveness of Frankia entrapped in polyacrylamide gel.

Four Frankia-Casuarina endosymbiont strains were tested for their infectivity and effectiveness on C. equisetifolia plants after 1 d, 3 and 6 months of Frankia storage at 7, 28 and 40 degrees C as liquid-cultures and polyacrylamide gel (PAG)-immobilized inocula. At lower temperature the number of nodules was the same or higher than control for liquid inocula except after 6 months of storage. For the PAG-entrapped Frankia lower numbers of nodules were recorded due to the use of high Frankia titers. In general, the results showed comparable plant dry mass, total nitrogen, root to shoot and nodules to plant ratios at lower temperature treatments. Increasing time and temperature was accompanied with reduced infectivity and effectiveness on inoculated plants. No nodulation was scored on plants inoculated with liquid and PAG-entrapped inocula stored at 40 degrees C for 6 months; subsequently, plant growth was inhibited. The variations in results obtained for different strains and treatments lead to variations in plant nitrogen-fixing ability. The use of PAG as a carrier for different Frankia strains is suggested; the recommended storage temperature for PAG-immobilized Frankia in 7-28 degrees C for up to 3 months.

Measurements of hyperpolarized gas properties in the lung. Part III: (3)He T(1).

Hyperpolarized (3)He spin-lattice relaxation was investigated in the guinea pig lung using spectroscopy and imaging techniques with a repetitive RF pulse series. T(1) was dominated by interactions with oxygen and was used to measure the alveolar O(2) partial pressure. In animals ventilated with a mixture of 79% (3)He and 21% O(2), T(1) dropped from 19.6 sec in vivo to 14.6 sec after cardiac arrest, reflecting the termination of the intrapulmonary gas exchange. The initial difference in oxygen concentration between inspired and alveolar air, and the temporal decay during apnea were related to functional parameters. Estimates of oxygen uptake were 29 +/- 11 mL min(-1) kg(-1) under normoxic conditions, and 9.0 +/- 2.0 mL min(-1) kg(-1) under hypoxic conditions. Cardiac output was estimated to be 400 +/- 160 mL min(-1) kg(-1). The functional residual capacity derived from spirometric magnetic resonance experiments varied with body mass between 5.4 +/- 0.3 mL and 10.7 +/- 1.1 mL. Magn Reson Med 45:421-430, 2001.

Effects of symbiosis with Frankia and arbuscular mycorrhizal fungus on the natural abundance of 15N in four species of Casuarina.

The effect of interactions between Casuarina species, Frankia strains and AMF on nitrogen isotope fractionation within the plant were determined under conditions where changes in source nitrogen were minimized by growing plants in mineral nitrogen-deficient conditions and without added organic N. Casuarina cunninghamiana, C. equisetifolia, C. glauca, and C. junghuniana were inoculated singly with three Frankia strains or were dual inoculated with Frankia and Glomus fasciculatum. The %N and delta 15N of separated parts of plants inoculated with the three Frankia strains or with Frankia + Glomus were not significantly different within Casuarina species. However, the slow-growing C. junghuniana differed in several variables from the other three species. There was a highly significant, linear relationship between the natural logarithms of cladode N content and delta 15N of plants of the four Casuarina species when inoculated with Frankia or with Frankia + Glomus, showing that nitrogen supply and the correlated variable, plant growth rate, were major determinants of delta 15N. Provision of small quantities of (NH4)2SO4 or KNO3 increased several-fold the growth of three of the Casuarina species when inoculated with Frankia alone or with Frankia + Glomus. Within species, mycorrhizal and non-mycorrhizal plants receiving supplementary soluble phosphate were of similar dry weights at harvest. delta 15N values for cladodes of C. cunninghamiana, C. equisetifolia and C. glauca were similar, but values for the poor growing C. junghuniana were more variable and, with the exception of plants receiving KNO3, were lower than those of the other three species. Reduced growth due to suboptimal availability of N or P had a major influence on delta 15N and, in these conditions where plants could not access significant amounts of organic N, outweighed any effects on cladode delta 15N of colonization by Glomus. delta 15N values of nodules were higher than other parts of Frankia or Frankia + Glomus inoculated Casuarinas, conceivably due to retention in nodules of fixed N, with delta 15N close to zero.

Magnetic resonance angiography with hyperpolarized 129Xe dissolved in a lipid emulsion.

Hyperpolarized (HP) 129Xe can be dissolved in biologically compatible lipid emulsions while maintaining sufficient polarization for in vivo vascular imaging. For xenon in Intralipid 30%, in vitro spectroscopy at 2 T yielded a chemical shift of 197 +/- 1 ppm with reference to xenon gas, a spin-lattice relaxation time T1 = 25.3 +/- 2.1 sec, and a T2* time constant of 37 +/- 5 msec. Angiograms of the abdominal and pelvic veins in the rat obtained with 129Xe MRI after intravenous injection of HP 129Xe/Intralipid 30% into the tail demonstrated signal-to-noise ratios between 8 and 29. An analysis of the inflow effect on time-of-flight images of two segments of the inferior vena cava yielded additional information. The mean blood flow velocity was 34.7 +/- 1.0 mm/sec between the junction of the caudal veins and the kidneys and 13.3 +/- 0.8 mm/sec at the position of the diaphragm. The mean volume flow rates in these segments were 7.2 +/- 3.4 ml/min and 11.0 +/- 2.8 ml/min, respectively. Intravenous delivery of HP 129Xe dissolved in a carrier may lead to novel biomedical applications of laser-polarized gases.

In vivo magnetic resonance vascular imaging using laser-polarized 3He microbubbles.

Laser-polarized gases (3He and 129Xe) are currently being used in magnetic resonance imaging as strong signal sources that can be safely introduced into the lung. Recently, researchers have been investigating other tissues using 129Xe. These studies use xenon dissolved in a carrier such as lipid vesicles or blood. Since helium is much less soluble than xenon in these materials, 3He has been used exclusively for imaging air spaces. However, considering that the signal of 3He is more than 10 times greater than that of 129Xe for presently attainable polarization levels, this work has focused on generating a method to introduce 3He into the vascular system. We addressed the low solubility issue by producing suspensions of 3He microbubbles. Here, we provide the first vascular images obtained with laser-polarized 3He. The potential increase in signal and absence of background should allow this technique to produce high-resolution angiographic images. In addition, quantitative measurements of blood flow velocity and tissue perfusion will be feasible.

Analysis of LMW RNA profiles of Frankia strains by staircase electrophoresis.

An optimized technique of polyacrylamide gel electrophoresis, Staircase Electrophoresis (SCE), was applied to determine the stable Low Molecular Weight RNA (LMW RNA) profiles of 25 Frankia strains from diverse geographic origins and host specificity groups as well as species from other actinomycete genera. Application of the technique permits the rapid identification of Frankia strains and their differentiation from other actinomycetes. The isolates used in this study were grouped in eight clusters, each comprising strains with identical LMW RNA profiles. Comparison of these results with others obtained from DNA sequences or DNA hybridization methods suggest a high degree of complexity in the genus Frankia. Application of SCE to profile LMW RNA should in the future facilitate biodiversity studies of Frankia and discrimination of new species.

Magnetic resonance microscopy of toxic renal injury induced by bromoethylamine in rats.

The alkylhalide 2-bromoethylamine hydrobromide (BEA) produces renal injury in rats that mimics analgesic-related renal injury in humans. Our purpose was to examine this injury, in vivo in rats, with magnetic resonance (MR) microscopy and correlate MR findings with findings from light microscopy of hematoxylin-eosin-stained sections. Rats (n = 48) were injected intravenously with BEA (150 mg/kg) or saline and imaged with MR 6, 48, and 336 hr later. The spin-spin relaxation time, T2, was measured from the cortex to the papilla. In other rats, we measured regional water content of the kidney. Renal injury was present 48 and 336 hr after BEA dosing based on increased renal organ weights, decreased urine specific gravity, and significant renal lesions (H & E). T2 was elevated in the inner stripe of the outer medulla in injured kidneys at 48 hr. The differences in T2 between cortex and outer medulla were also elevated 48 hr after BEA. In the inner medulla, there were no changes in T2 after BEA treatment. However, in all groups there were significant regional differences in T2. The value of T2 increased from outer to inner medulla and this gradient was directly correlated with water content. Thus, MR microscopy detected damage in the outer medulla after BEA injury but not the damage in the inner medulla. T2 appeared to reflect the water content in the different regions of the medulla. The noninvasive in vivo capability of MR microscopy, with its high sensitivity to tissue water, allows the toxicologist to monitor the progression and regression of toxic insult in the same animal. At present the technology is complicated. The precise and accurate measure of MR-sensitive parameters in live animals at microscopic resolution is difficult. However, as the technology matures, there will be significant improvements providing the toxicologist a unique in vivo tool.

Extravascular toxicity of two magnetic resonance contrast agents. Preliminary experience in the rat.

We compared the relative toxicities of standard concentrations of two magnetic resonance imaging (MRI) contrast agents, ionic gadolinium diethylenetriaminepentacetic acid (DTPA) and low-osmolar gadolinium-1, 4, 7 tris (carboxymethyl)-10-(2'-hydroxypropyl)-1, 4, 7, 10 tetra-azacyclododecane (HP-DO3A) with that of the conventional radiographic contrast medium meglumine diatrizoate, when extravasated into the deep dermal tissues of laboratory rats. Gadolinium-DTPA caused moderate necrosis, hemorrhage, and edema which was not statistically different than meglumine diatrizoate. In contrast, gadolinium HP-DO3A was significantly less toxic than meglumine diatrizoate. Additional experience will be needed in order to determine whether these laboratory results will be clinically relevant in humans.

Extravascular extravasation of radiographic contrast media. Effects of conventional and low-osmolar agents in the rat thigh.

We compared the damage resulting from intradermal injection of four commonly used radiographic contrast media in laboratory rats. Sixty percent meglumine diatrizoate (Reno M 60) and ioxaglate (Hexabrix) produced significantly more ulceration and crusting on gross inspection and more necrosis, edema, and hemorrhage on histologic evaluation than iopamidol 300 (Isovue) or 0.9% (normal) saline. Thirty percent meglumine diatrizoate (Reno M Dip) had an intermediate toxicity, resulting in significantly more visible swelling and more microscopically detected hemorrhage than iopamidol or saline, but less ulceration/crusting and necrosis than Reno M 60 and ioxaglate. Since the three contrast agents of similar osmolality produced different degrees of tissue damage, our results suggest that factors other than high osmolality are partially responsible for determining the severity of injuries from extravasated contrast media.

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots.

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA1 and GA4. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA9- as well as GA20-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod (-) and fix (-) mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli.

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with (3)H-, (14)C- and (2)H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [(3)H]tryptophan nor did cultures convert [(14)C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.

ABSCISIC ACID AND DORMANCY IN ROOT-NODULATED ALNUS GLUTINOSA (L.) GAERTN.

Treatment of three-month or six-month-old water culture plants of Alnus glutinosa with 0.1 mol m-3 (±) abscisic acid (ABA) induced formation of resting buds after 30 to 60 d. Leaf dry weight per plant decreased by 40 to 45 % during this period but root and nodule dry weights were unchanged relative to control plants. [2-14 C] ABA was taken up by the root system within 5 d and after 30 or 60 d the ABA content of plants fed 01 mol m-3 ABA was 60 and 119 times that of control plants. In control plants, the levels of 'free'cis(Z) ABA were similar to or exceeded 'free'trans(E) ABA in all plant parts while, in general, the converse was true for these isomers in 'bound' form. In ABA-treated plants, all isomeric forms of ABA were present at much higher amounts than in the controls but, whereas in leaves and shoot apices the amount of cis ABA in both 'free' and 'bound' forms was much greater than the trans isomer, in roots and nodules trans ABA was the dominant isomer in both 'bound' and 'free' forms. Most biologically active 'free'cis ABA thus accumulated in the leaves and shoot apex, where effects of ABA treatment on growth were most evident, whereas in roots and nodules where biologically inactive 'free' and 'bound'trans ABA were dominant, growth was relatively unaffected. Amounts of 'free' ABA in nodules were several times those recorded previously in nodules of dormant plants, in keeping with earlier suggestions that ABA is unlikely to have a precise regulatory role in growth and dormancy of nodules.

WINTER FIXATION OF NITROGEN IN SCOTCH BROOM (CYTISUS SCOPARIUS L.).

The retention of green lateral branches by Scotch Broom allows nitrogen fixation to continue during mild winters. Tolerance of low soil temperatures is suggested to be a major factor contributing to the occurrence of winter nitrogen fixation.