Iron-Chelating Compounds Produced by Soil Pseudomonads: Correlation with Fungal Growth Inhibition

Strains of Pseudomonas putida, Pseudomonas sp., and Pseudomonas aeruginosa were examined for their ability to grow in the presence of the iron chelator, ethylenediamine-di-(o-hydroxyphenylacetic acid). In vitro fungal inhibition assays showed that the isolates varied in their ability to inhibit the growth of representative fungal plant pathogens. Fungal inhibition in vitro was superior to that of previously reported Pseudomonas sp. Studies with Fusarium oxysporum forma sp. lycopersici and a susceptible tomato cultivar demonstrated that Pseudomonas putida PPU3.1 was able to significantly reduce wilt disease.     

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.     

Specificity of the Na+-dependent monocarboxylic acid transport pathway in rabbit renal brush border membranes

The substrate specificity of a Na+-dependent transport pathway for L-lactate was studied in rabbit renal brush border membrane vesicles. Jmax for L-lactate transport was unaffected by the presence of a fixed concentration of two different short-chain monocarboxylic acids, while the apparent Kt(Ka) for L-lactate increased, and this is compatible with competitive inhibition. The inhibitor constants ("Ki"'s) for the transport pathway for the two solutes examined closely corresponded to the respective "Ki"'s derived from a Dixon plot. A broad range of compounds were then tested as potential inhibitors of L-lactate transport, and the "Ki"'s thereby derived yielded specific information regarding optimal substrate recognition by the carrier. A single carboxyl group is an absolute requirement for recognition, and preference is given to 3 to 6 C chain molecules. Addition of ketone, hydroxyl and, particularly, amine groups at any carbon position, diminishes substrate-carrier interaction. Intramolecular forces, notably the inductive effects of halogens, may play a role in enhancing substrate-carrier interaction; however, no correlation was found between pKa and "Ki" for the substrates examined. We conclude that a separate monocarboxylic acid transport pathway, discrete from either the D-glucose, alpha or beta neutral amino-acid, or dicarboxylic acid carriers, exists in the renal brush border, and this handles a broad range of monocarboxylates.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Embryo transfer in water buffalo (Bubalus bubalis)

A normal, live 35-kg water buffalo bull calf was born 300 days after it was nonsurgically collected as a 7-day blastocyst from a water buffalo donor and transferred nonsurgically to an unrelated water buffalo recipient. The development of estrus synchronization, superovulation and estrus detection methods in water buffalo are described.     

1H nmr studies of complexes of ferric leghemoglobin with substituted pyridines and nicotinic acids

The ¹H nuclear magnetic resonance (nmr) spectra of complexes of soybean ferric leghemoglobin with 3-substituted pyridines and 5-substituted nicotinic acids have been recorded in order to determine the influence of axial ligands on heme electronic structure. The hyperfine shifted resonances of the heme group were assigned by analogy to previous assignments for the pyridine and nicotinic acid complexes of leghemoglobin. The spectra are characteristic of predominantly low-spin ferric heme complexes. For the pyridine complexes, the rate of ligand exchange was found to increase with decreasing ligand pK_A. For many of the complexes, optical and nmr spectra reveal the presence of an equilibrium mixture of high- and low-spin states of the iron atom. The percentage of high-spin component increases with decreasing ligand pK_A Smaller hyperfine shifts are noted for leghemoglobin complexes with ligands capable of weak ligand → metal π bonding. The pattern of hyperfine shifted resonances is similar for all complexes studied and indicates that the overall heme electronic structure is dominated by the bonding to the proximal histidine.     

High-performance liquid chromatography of coproporphyrin isomers.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.     

Use of the F test for determining the degree of enzyme-kinetic and ligand-binding data. A Monte Carlo simulation study

1. Initial-rate data were simulated for 13 representative enzyme mechanisms with the use of several distributions of rate constants in order to locate conditions leading to v([S]) curves in physiological ranges of substrate concentration. 2. In all, 420 sets of such v([S]) curves were generated with the use of several choices of substrate concentration range (two, three or four orders of magnitude), number of experimental points (10, 15 or 20), error on v (5-10%) and standard deviation on v (5-9%) in order to simulate experimental results in a number of possible ways. 3. Curve-fitting was carried out to rational functions of degree 1:1, 2:2, . . ., 5:5 until there was no statistically significant decrease in the sum of weighted squared residuals as judged by the F test at 95% and 99% confidence levels. 4. It was checked whether the non-linear regression program had located a good minimum in the sum of squares by also fitting the data with the correct values of parameters as starting estimates. 5. A similar procedure was adopted with 110 sets of binding data simulated for 11 models, and the F test was used to see if fractional-saturation data generated by a binding polynomial of order n could be adequately fitted by one of order m, m less than n. 6. From the 530 simulations the F test was successful in fixing the correct degree with a probability of 0.62 at the 95% confidence level, but this fell with increase in degree as follows: 1:1 (0.98), 2:2 (0.71), 3:3 (0.43) and 4:4 (0.34), the first numbers being the degree of the rate equation and those in parentheses referring to the 95% confidence level. 7. It made little difference whether the 95% or the 99% confidence level was consulted, as there were very few borderline cases. 8. The chance of detecting deviations from Michaelis-Menten kinetics, i.e. terms of at least second-order in a rate equation of degree n:n, n greater than 1, was estimated to be about 0.8. 9. The probability of the F test leading to a spurious result due to error in the data was found to be about 0.04. 10. The probability with which 4:4 mechanisms can lead to v([S]) plots with no, one, two or three turning points was computed, and it was established that there is a small but finite chance that the increase in degree that occurs in some mechanisms when ES in equilibrium EP interconversions are explicitly allowed for can be detected by the F test.     

The sequence GGCmCGG is resistant to MspI cleavage.

MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MspI rather than a novel form of DNA methylation at this site in mammalian cells.