Microplate Assay for Colletotrichum Spore Production

A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs.     

Characterization of specific fluorenylmethyloxycarbonyl-containing calmodulin adducts by spectroscopy and phosphodiesterase stimulation

A reagent (I, N⁴-(9-fluorenylmethyloxycarbonyl-4-amino-1-oxyl-4-succinimidyloxycarbonyl-2,2,6,6-tetramethylpiperidine)) that acylates calmodulin specifically at lysines 75 and 148 was recently described (Jackson and Puett, 1984). Chromatographic procedures are described that permit purification to apparent homogeneity of a 1 : 1 and a 2 : 1 adduct characterized by modification at just Lys 75 or at Lys 75 and Lys 148, respectively. These adducts are suitable for detailed characterization in an effort to provide information on calmodulin structure-function relationships. The adducts were incapable of, or exhibited low potency (e.g., 0.1% that of calmodulin) in, stimulating the activity of an activatable bovine brain cyclic nucleotide phosphodiesterase (3,5-cyclic AMP 5-nucleotidehydrolase, EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotational correlation times of approximately 3–6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (5–7 nsec). Thus, the nitroxide reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestingly, removal of the fluorenylmethyloxycarbonyl portion from the 1 : 1 adduct to give a deprotected 1 : 1 adduct resulted in apparent greater mobility of the probe, since the rotational correlation coefficient was found to be 1 nsec. Circular dichroic spectra were obtained over the wavelength interval 200–250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmodulin, exhibited a Ca²⁺-mediated increase in helicity, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca²⁺ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetic resonance spectra were determined in the aromatic region (6–8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide with ascorbate. The nitroxide had little effect on the chemical shifts of the two tyrosines and the single histidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound II, -N-(9-fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct at Lys 75 and a 2 : 1 adduct at Lys 75 and Lys 148. Proton NMR spectra of adducts with II were not complicated by the relaxation effects arising from adducts with I; thus more definitive assignments could be made to the upfield resonances, including the fluorene protons. Again, it was possible to conclude that adduct formation had no major effect on the tertiary structure of the protein as monitored by chemical shifts associated with various residues. We conclude that modification of just Lys 75, a residue in the long connecting helix of calmodulin, does not lead to major changes in protein conformation but does interfere with the ability of calmodulin to stimulate an activatable form of bovine brain cyclic nucleotide phosphodiesterase.     

Formation of acetylcarnitine in muscle of horse during high intensity exercise

To study the changes in carnitine in muscle with spring exercise, two Thoroughbred horses performed two treadmill exercise tests. Biopsies of the middle gluteal were taken before, after exercise and after 12 min recovery. Resting mean muscle total carnitine content was 29.5 mmol.kg-1 dry muscle (d.m.). Approximately 88% was free carnitine, 7% acetylcarnitine and acylcarnitine was estimated at 5%. Exercise did not affect total carnitine, but resulted in a marked fall in free carnitine and almost equivalent rise in acetylcarnitine. The results are consistent with a role for carnitine in the regulation of the acetyl-CoA/CoA ratio during sprint exercise in the Thoroughbred horse by buffering excess production of acetyl units.     

Effect on in Vitro Pollen Growth of an Isolated Style Glycoprotein Associated with Self-Incompatibility in Nicotiana alata

The effect on in vitro pollen tube growth of an isolated style glycoprotein (S₂-glycoprotein) associated with self-incompatibility in Nicotiana alata was investigated. Tube growth of pollen bearing the S₂-allele was inhibited, but tube growth of pollen bearing other alleles was not affected. Inhibition showed a dose response effect. The percentage of pollen grains that germinated was not significantly affected by the S₂-glycoprotein. Growth of S₂-pollen in the presence of the S₂-glycoprotein resulted in increased binding to the pollen of monoclonal antibody (PCBC3) which has a primary specificity for α-l-arabinofuranosyl residues. Growth of pollen bearing other alleles in the presence of the glycoprotein resulted in no increased binding of the antibody.     

Developmental studies on creatine kinase. Isoenzyme in rat gastrointestinal tract

Creatine kinase activity and its isoenzymatic profile in rat intestinal mucose during normal development have been studied. Creatine kinase enzymatic activity increased stepwise during fetal development and the first week of life. An isoenzymatic pattern of exclusively CK-BB types occurred in all segments of the digestive tract during the early fetal stage. The isoenzyme profile of creatine kinase in the esophagic tissue with advancing maturation of the fetus shifted in the same way as in adults, with preferential concentration of CK-MM. However, CK-BB continued to be the main isoenzyme in the rest of the digestive tract. Our results show that rats are particularly suitable for experimental studies of intestinal creatine kinase isoenzymes.