Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

A toxin fromPasteurella multocida serogroup D enhances swine herpesvirus 1 replication/lethality in vitro and in vivo

In swine, the nasal turbinate epithelium is both a site of swine herpesvirus 1 (pseudorabies virus, PRV) replication and a tissue affected by toxin fromPasteurella multocida serogroup D. We examined the effects of exposure to PRV and exposure to toxin in mice, swine, and nasal turbinate cell cultures. Increased mortality in mice was observed when nonlethal doses of PRV (1000 or 100 plaque-forming units, PFU) were administered along with nonlethal doses (60–200 ng/kg) of toxin. In swine, clinical disease and death in adult pigs was observed after an intradermal injection of toxin (20 ng/kg) and intranasal exposure to 1000 PFU/kg of PRV. Nasal turbinate cell cultures incubated with toxin and PRV had increased protein synthesis, DNA synthesis, and increased recovery of virus particles. These findings indicate that a toxin fromP. multocida serogroup D enhances swine herpesvirus 1 replication and lethality in cell cultures and animal models.     

Isolation ofAcholeplasma oculi from human amniotic fluid in early pregnancy

Acholeplasmas have been isolated from a variety of animals, insects, and plants, but onlyAcholeplasma laidlawii has previously been found in humans. We have isolatedAcholeplasma oculi in pure culture from the amniotic fluid of a woman at 19 weeks of gestation. The organism was positively identified by growth inhibition, epi-immunofluorescence, and arbutin hydrolysis. Demonstration of organisms directly in amniotic fluid by DNA fluorochrome and immunofluorescence staining provided additional evidence that the isolate was genuine and not a medium contaminant. The remainder of the pregnancy was unremarkable, and a full-term male infant was delivered without complications. Even though there is some evidence possibly associatingA. oculi with various diseases in livestock, the prevalence and significance ofA. oculi in humans has not been determined.     

Strategies for the use of computational SAR methods in assessing genotoxicity

The relationship between computational SAR studies and relevant data gathering and generation activities is complex. First, the chemical class to be studied is selected on the basis of information requirements for hazard identification and assessment. Membership in the class is determined by consideration of chemical structure and reactivity. Compilation of the existing bioassay data for this chemical class follows immediately from the specification of the class. Bioassay data, qualitative knowledge of general chemical reactivities in this class, and knowledge concerning potential interactions with biomolecular targets all contribute to the derivation of possible mechanisms for biological activity. Computational studies based on modeling the proposed mechanism of action and/or the existing data base can provide a quantitative basis for the differentiation between chemicals. There is the opportunity for continuing feedback between the quantitative computational studies and the development of a relevant bioassay data base for this chemical class. The qualitative and quantitative information on the potential biological responses obtained will provide a rational basis for extrapolation from the extant data base to the chemicals of interest, and to biological responses significant to the assessment for which complete data are unavailable. Knowledge concerning possible mechanisms of action and preexisting data determine the type of computational study that will be most useful.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Subcellular distribution,molecular dynamics and catabolism of plasmalogens in myocardium

Recent studies have implicated accelerated sarcolemmal phospholipid catabolism as a mediator of the lethal sequelae of atherosclerotic heart disease. We have demonstrated that plasmalogens are the predominant phospholipid constituents of canine myocardium and that plasmalogens are hydrolyzed by a novel calcium independent plasmalogen selective phospholipase A2. Since the activities of phospholipases are modulated by the molecular dynamics and interfacial characteristics of their phospholipid substrates, we compared the molecular dynamics of plasmenylcholine and phosphatidylcholine vesicles by electron spin resonance spectroscopy and deuterium magnetic resonance spectroscopy. Plasmenylcholine vesicles have separate and distinct molecular dynamics in comparisons to their phosphatidylcholine counterparts as ascertained by substantial decreases in the angular fluctuations and motional velocities of probes attached to their sn-2 aliphatic constituents. Furthermore, since free radical oxidation of myocardial lipid constituents occurs during myocardial ischemia and reperfusion, we demonstrated that ¹O₂ mediated oxidation of plasmenylcholine resulted in the generation of several products which have chromatographic characteristics and molecular masses corresponding to 2-acyl lysophosphatide derivatives. Taken together, these studies underscore the biologic significance of the predominance of sarcolemmal plasmalogens present in mammalian myocardium and suggest that their catabolism by plasmalogen selective phospholipases and/or oxidative processes may contribute to the lethal sequelae of myocardial ischemia.     

Fungispecificity of fluconazole against Candida albicans

Candida albicans is an opportunistic pathogen of human mucosal surfaces. Colonization of oral and vaginal mucosa by this yeast is antagonized by the resident normal bacterial population. However, antibacterial therapy can alter the normal flora to allow fungal cells to attach, grow and invade host tissues. We studied the antimicrobic activity of fluconazole against clinical isolates of oral and vaginal bacteria and Candida albicans in vitro and in vivo by scanning and transmission electron microscopy; we also compared the bactericidal activity of fluconazole with clotrimazole in vitro by microbiologie assay. Fluconazole lysed fungi but did not change the ultrastructure of bacteria. Clotrimazole, but not fluconazole, was bactericidal against lactobacillus and streptococcus, the principal species of the oral and vaginal cavities. We conclude that Candida albicans, but not oral and vaginal bacteria, is susceptible to fluconazole. These observations help explain the antimycotic specificity of fluconazole and its efficacy against candidiasis in humans.     

Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca²⁺. It has a K_i of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca²⁺-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD cholesterylphosphoryldimethylethanolamine     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.