Properties of protoplasts from the thermophile Bacillus coagulans and their significance for genetic studies

Regeneration of protoplasts of Bacillus coagulans was optimized by using low lysozyme concentrations and glycerol as the osmotic support. Protoplasts formed from cells grown at higher temperatures were thermostable and capable of regeneration at 55°C. Transfer of plasmids pAB224 and pUB110, using either whole cells or protoplast transformation was not achieved, despite using a variety of conditions. However, plasmid transfer was achieved by fusion with B. subtilis protoplasts containing plasmid pAB224.     

Improved medium for recovery and enumeration of the farmer's lung organism, Saccharomonospora viridis.

A new medium, which we propose to call R8, was developed for the isolation and enumeration of the thermophilic actinomycete, Saccharomonospora viridis. This organism has been implicated in a range of hypersensitivity pneumonitides, including farmer's lung, but is generally isolated in small numbers from contaminated environments. Recovery of S. viridis from moldy hay and mushroom compost on R8 medium was compared with recovery on conventional media. S. viridis was isolated from both substrates but in highest numbers and most consistently on the R8 medium. The selectivity of this medium was best observed when the sedimentation chamber method was used for hay samples. Here S. viridis accounted for up to 80% of the total number of actinomycetes recovered on R8 and could not be recovered on rifampin selective medium under the same conditions. R8 was also found to be an efficient recovery medium for a range of thermophilic actinomycetes from mushroom compost and for another allergenic species, Faenia rectivirgula, from moldy hay. Contamination of isolation plates by thermophilic bacilli was reduced on R8 compared with the activity on half-strength tryptone soy agar, supplemented with 0.2% casein hydrolysate, and this, together with specific improvements in S. viridis growth, accounts for the selective effect. It is possible that the occurrence of S. viridis and its role as a causative agent of hypersensitivity pnuemonitis have been underestimated by the use of suboptimal recovery protocols. It is hoped that use of R8 in conjunction with dilution plate techniques will generate information on the ecology of S. viridis and contribute to health risk assessment studies.     

Effects of Metals on a Range of Streptomyces Species

A plate diffusion method was used to assess the tolerance of 28 mesophilic and 4 thermophilic species of streptomycetes to eight metals. This method allowed a qualitative screen of the effects of the metals on growth, on sporulation, and in some cases, on secondary metabolite production. Tolerant and sensitive species were identified, some of which exhibited the same response (i.e., tolerance or sensitivity) to a number of metals. Experiments in liquid culture were used to quantify the precise concentration ranges of the metals at which the most tolerant or sensitive species could grow. Distribution profiles of the sensitivities of all species examined toward different metals revealed that the order of toxicity was Hg > Cd > Co > Zn > Ni > Cu > Cr > Mn.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

Light-evoked walking in crayfish: Behavioral and neuronal responses triggered by the caudal photoreceptor

The caudal photoreceptors (CPRs) of crayfish (Procambarus clarkii) can trigger walking and abdominal movements by their response to light.

Deletion mapping of the medulloblastoma locus on chromosome 17p

Isochromosome 17q has previously been observed consistently in cytogenetic studies of medulloblastoma, the most common posterior fossa neoplasm in children. We performed a restriction fragment length polymorphism (RFLP) investigation of medulloblastoma which showed a loss of chromosome 17p sequences in 45% of these tumors. This finding was predictive of a poor clinical response to treatment. A contiguous panel of markers permitted mapping of the deletion to 17p12-p13.1, the same chromosomal region for which loss of alleles has been shown in tumor specimens from patients with colon cancer, and the same region to which the p53 gene has been mapped. This suggests that medulloblastoma is associated with a recessive oncogene on chromosome 17p that may be involved in the genesis of several embryologically unrelated neoplasms and that the absence of this gene in tumor tissue has prognostic significance.     

Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.