Special-purpose legume genetic resources conserved for agricultural, industrial, and pharmaceutical use

To help feed, clothe, house, improve land productivity, confront difficult resource necessities, and stabilize ravaged land resources through reforestation, there is need for a revitalized worldwide assessment of little-known legume species. Many legumes produce economically important organic compounds such as gums, dyes, Pharmaceuticals, pesticides, phytochemicals, and many specialty products. However, most legumes have never been or have just begun to be studied for phy-tochemical or biologically active components, and new reservoirs of commercially valuable materials remain to be discovered. Not only could these legumes provide healthier and a more secure food supply for Third World countries, but they can protect fragile soil resources where erosion now threatens tragedy.     

Evaluation of media for recovery of aerosolized bacteria

Disease transmission by airborne bacteria is well known. Bacterial burden in indoor air is estimated by sampling the air and estimating Colony Forming Units (CFU) using a variety of media. In this study, the recovery of bacteria, after aerosolization in an aerosol chamber, and employing a variety of media, was compared to that achieved using Tryptic Soy Agar medium. The total number of cells present was determined by direct microscopy. All trials were conducted at approximately the same relative humidity (RH) and temperature using the same collection device. Twelve species of bacteria were tested and a total of 120 media or media combinations were evaluated. Recovery on 64 media formulations was significantly lower for all strains examined, and therefore, excluded from further consideration for the purposes of this study. Data for 56 of the media are presented. Three species (Bacillus subtilis, Staphylococcus aureus andSerratia marcescens) were selected as representative for reporting and testing recovery success. It is concluded that, for the media included in the study, there are large differences in recovery and successful recovery is related both to the effect of aerosolization and the type of medium employed for recovery. Brain Heart Infusion Agar (with horse serum), Tryptic Soy Agar and Mueller Hinton Agar yielded the best recoveries of aerosolized cultures. The most important finding was that only a small fraction of the airborne bacterial populations, enumerated by direct microscopy, could be recovered on any of the media tested, suggesting that culturable bacterial count is not a satisfactory means of estimating air microbial pollution.     

Immunization with Temperature-Sensitive Mutants of Actinobacillus pleuropneumoniae Induces Protective Hemolysin-Neutralizing Antibodies in Mice

The immunogenicity and protective potential of three temperature-sensitive mutants of Actinobacillus pleuropneumoniae were evaluated in mice with respect to antibodies against the capsular polysaccharide, lipopolysaccharide, outer membrane proteins, and hemolysin protein. Antibodies to the capsular polysaccharide and lipopolysaccharide could not be correlated with protection in the mice; there were no significant differences among the anti-capsular and anti-lipopolysaccharide antibody titers regardless of the severity of infection. Sera from mice immunized with the mutants and challenged with the wild type contained antibodies that reacted in immunoblots to four major outer membrane proteins (66, 39, 29, and 16 kDa) regardless of the severity of infection after challenge. Both the tight and coaster mutants synthesized and secreted the 105-kDa hemolysin protein exotoxin in vitro and in vivo; hemolysin protein neutralization titers and the blotting intensity of the sera, however, varied inversely with the severity of infection. Sera from mice surviving challenge with little to no lung involvement stained the hemolysin band more intensely and had significantly higher neutralization titers (P < 0.05) than sera from mice that either died or survived with severe pulmonary hemorrhage. These results confirm the importance of the hemolysin in pathogenesis and the need for including it in any vaccine preparation. Received: 26 August 1996 / Accepted: 3 September 1996     

Spectroscopic characterization of quinone-site mutants of the bacterial photosynthetic reaction center

Site-specific mutations in the quinone binding sites of the photosynthetic reaction center (RC) protein complexes of Rhodobacter (R.) capsulatus caused pronounced effects on sequential electron transfer. Conserved residues that break the twofold symmetry in this region of the RC – M246Ala and M247Ala in the QA binding pocket, and L212Glu and L213Asp in the QB binding pocket – were targeted. We constructed a QB-site mutant, L212Glu-L213Asp Ala-Ala, and a QA-site mutant, M246Ala–M247Ala Glu-Asp, to partially balance the differences in charge distribution normally found between the two quinone binding sites. In addition, two photocompetent revertants were isolated from the photosynthetically-incompetent M246Glu-M247Asp mutant: M246Ala–M247Asp and M246Gly–M247Asp. Sequential electron transfer was investigated by continuous light excitation and time-resolved electron paramagnetic resonance (EPR), and time-resolved optical techniques. Several lines of EPR evidence suggested that the forward electron transfer rate to QA, kQ, was slowed in those strains containing altered QA sites. The slower rates of secondary electron transfer were confirmed by time-resolved optical results with the M246Glu-M247Asp mutations in the QA site resulting in a dramatically lowered secondary electron transfer efficiency [kQ < (2 ns)-1] in comparison with either the native R. capsulatus RC or the QB site mutant [kQ (200 ps)-1]. Secondary electron transfer in the two revertants was intermediate between that of the native RC and the QA mutant. The P+ QA- PQA charge recombination rates were also changed in the strains that carried altered QA sites. We show that local mutations in the QA site, presumably through local electrostatic changes, significantly alter binding and electron transfer properties of QA.     

Muon Spectroscopy Applied to Biological Systems: A Study of Thiyl Radicals, RS

Thiyl radicals (RS.) are formed when positive muons (μ⁺) are implanted into solutions of thiocarbonyl compounds (C = S) in ethanol, tetrahydrofuran or formamide solvents. A solvent dependence is found, which reflects changing electronic interactions and overall conformations, suggesting that the properties of RS. radicals may be dependent on the polarity (philicity) of the environment in which they are formed.     

Mutations in the heavy chain of cytoplasmic dynein suppress the nudF nuclear migration mutation of Aspergillus nidulans

To identify proteins that interact directly or indirectly with the NUDF protein, which is required for nuclear migration in Aspergillus nidulans, we initiated a screen for extragenic suppressors of the heat-sensitive nudF6 mutation. Suppressor mutations in at least five genes, designated snfA–snfE, caused improved growth and nuclear migration at high temperatures compared to the nudF6 parent. Two snfC mutations mapped near the nudA gene, which encodes the cytoplasmic dynein heavy chain, and could be repaired by transformation with wild-type nudA DNA, demonstrating that they are mutations in nudA. The snfC mutations are bypass suppressors of nudF and genetic evidence indicated that NUDA and NUDF act in the same nuclear migration pathway. Taken together, our data suggests that NUDF affects nuclear migration by acting on the dynein motor system.     

Measurement of immunoglobulin synthesis using the ELISPOT assay

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.     

Ventrolateral medullary respiratory network participation in the expiration reflex in the cat.

The expiration reflex is a distinct airway defensive response characterized by a brief, intense expiratory effort and coordinated adduction and abduction of the laryngeal folds. This study addressed the hypothesis that the ventrolateral medullary respiratory network participates in the reflex. Extracellular neuron activity was recorded with microelectrode arrays in decerebrated, neuromuscular-blocked, ventilated cats. In 32 recordings (17 cats), 232 neurons were monitored in the rostral (including B?tzinger and pre-B?tzinger complexes) and caudal ventral respiratory group. Neurons were classified by firing pattern, evaluated for spinal projections, functional associations with recurrent laryngeal and lumbar nerves, and firing rate changes during brief, large increases in lumbar motor nerve discharge (fictive expiration reflex, FER) elicited during mechanical stimulation of the vocal folds. Two hundred eight neurons were respiratory modulated, and 24 were nonrespiratory; 104 of the respiratory and 6 of the nonrespiratory-modulated neurons had altered peak firing rates during the FER. Increased firing rates of bulbospinal neurons and expiratory laryngeal premotor and motoneurons during the expiratory burst of FER were accompanied by changes in the firing patterns of putative propriobulbar neurons proposed to participate in the eupneic respiratory network. The results support the hypothesis that elements of the rostral and caudal ventral respiratory groups participate in generating and shaping the motor output of the FER. A model is proposed for the participation of the respiratory network in the expiration reflex.     

Response of avian embryonic brain to spatially segmented x-ray microbeams.

Duck embryo was studied as a model for assessing the effects of microbeam radiation therapy (MRT) on the human infant brain. Because of the high risk of radiation-induced disruption of the developmental process in the immature brain, conventional wide-beam radiotherapy of brain tumors is seldom carried out in infants under the age of three. Other types of treatment for pediatric brain tumors are frequently ineffective. Recent findings from studies in Grenoble on the brain of suckling rats indicate that MRT could be of benefit for the treatment of early childhood tumors. In our studies, duck embryos were irradiated at 3-4 days prior to hatching. Irradiation was carried out using a single exposure of synchrotron-generated X-rays, either in the form of parallel microplanar beams (microbeams), or as non-segmented broad beam. The individual microplanar beams had a width of 27 microm and height of 11 mm, and a center-to-center spacing of 100 microm. Doses to the exposed areas of embryo brain were 40, 80, 160 and 450 Gy (in-slice dose) for the microbeam, and 6, 12 and 18 Gy for the broad beam. The biological end point employed in the study was ataxia. This neurological symptom of radiation damage to the brain developed within 75 days of hatching. Histopathological analysis of brain tissue did not reveal any radiation induced lesions for microbeam doses of 40-160 Gy (in-slice), although some incidences of ataxia were observed in that dose group. However, severe brain lesions did occur in animals in the 450 Gy microbeam dose groups, and mild lesions in the 18 Gy broad beam dose group. These results indicate that embryonic duck brain has an appreciably higher tolerance to the microbeam modality, as compared to the broad beam modality. When the microbeam dose was normalized to the full volume of the irradiated tissue. i.e., the dose averaged over microbeams and the space between the microbeams, brain tolerance was estimated to be about three times higher to microbeam irradiation as compared with broad beam irradiation.