Isolation of Different Agrobacterium Biovars from a Natural Oak Savanna and Tallgrass Prairie

Populations of agrobacteria in excess of 10⁵ CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.     

Complementary Methodologies To Identify Specific Agrobacterium Strains

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.     

Pathophysiology of pH and Ca2+ in bloodstream and brain

The highlights of the literature and our work on tetany and hyperventilation are reviewed. Our studies concern the following: (1) the changes of [Ca2+] in circulating plasma caused by respiratory and "metabolic" acidosis and alkalosis; (2) critical plasma [Ca2+] levels associated with signs of tetany and neuromuscular blockade; (3) changes in cerebral [Ca2+]o caused by hypo- and hyper-calcaemia, and the changes in cerebral [Ca2+]o and pHo caused by acute systemic acidosis and alkalosis; and (4) effects of changing [Ca2+]o and pHo levels on synaptic transmission in hippocampal formation. Our main conclusions are (1) changes of plasma [Ca2+] caused by "metabolic" pH changes are greater than those associated with varying CO2 concentration; (2) acute systemic [Ca2+] changes are associated with small cerebral [Ca2+]o changes; (3) the decreases in systemic and cerebral [Ca2+]o caused by hyperventilation are too small to account for the signs and symptoms of hypocapnic tetany; (4) moderate decrease of [Ca2+]o depresses and its increase enhances synaptic transmission in hippocampal formation; and (5) H+ ions in extracellular fluid have a weak depressant effect on neuronal excitability. CO2 is a strong depressant, which is only partly explained by the acidity of its solution. CO2 concentration is a significant factor in controlling cerebral function.     

In vivo cytogenetic effects of cooked food mutagens

Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat. C57Bl/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells. PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow. In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested. PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood. However, dose responses were not observed. With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.     

Increased protoplast yield from oat leaves and bean internodes by non-injurious mechanical perturbation

Summary A simple new technique has been developed to greatly increase the yield of protoplasts from plant organs without injury to the plant. Mechanical perturbation (MP) by non-stressful rubbing of oat leaf segments and bean internodes yielded ten to twenty times more viable protoplasts than did controls. The increase in protoplast yield due to MP is best manifested, if the organs are excised and transferred to the cellulytic enzymes immediately after MP is given to the intact organ. The enzymes begin digesting from the lower end of the bean internodes and proceed acropetally. Vacuum infiltration of control oat leaf segments for 15 min with enzyme solution resulted in increased yield but less than due to MP. Increased levels of calcium (10 mM) in the medium decreased the yield of protoplasts from both control and MP-treated plant organs. EGTA significantly increased the yield of protoplasts from control oat leaf segments and marginally over that found in the control bean internodes. Cycloheximide increased the yield of protoplasts from oat leaf segments but not from bean internodes. It is suggested that MP may increase the susceptibility of cell wall polymers to cellulytic enzymes by reducing calcium cross linking. MP is thus a tool for increasing the yield of protoplasts from plant organs without causing injury.Abbreviations CHI cycloheximide - EGTA ethyleneglycol-bis-(ß-aminoethyl ether)-N,N-tetraacetic acid - FDA fluorescein diacetate - MP mechanical perturbation     

The effect of pregnancy and foaling on intravaginal pressure in pony mares

Pressures were recorded monthly at two sites in the vagina of each of five pregnant and five nonpregnant pony mares; pressures in five mares were also recorded weekly after foaling. The developing pregnancy did not influence pressure, and after foaling the integrity of the seal formed by the caudal reproductive tract was rapidly restored.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Metallothionein induction by nonsteroidal antiinflammatory drugs

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).     

State transitions,photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F relative fluorescence intensity at emission wavelength nm - F _o fluorescence intensity when all PS II traps are open - light 1 light absorbed preferentially by PS I - light 2 light absorbed preferentially by PS II - PBS phycobilisome - PS photosystem     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday