Epidermal growth factor (EGF) affects sulphydryl and disulphide levels in cultured mouse skin: possible relationship between effects of EGF and of the tabby gene on thiols

Studies from our laboratory have previously shown that the syndrome produced in the mouse by the X-linked gene tabby (Ta) has many features in common with human X-linked hypohidrotic ectodermal dysplasia. We have also demonstrated that tabby has abnormally elevated epidermal sulphydryl (SH): disulphide (SS) ratios, in common with an autosomal form of ectodermal dysplasia. The organs and tissues affected in many of the traits of these syndromes are targets of epidermal growth factor (EGF) and we have shown that the EGF-producing cells are deficient in tabby. In the present study we examined whether EGF affects SH and SS levels in normal mouse skin in tissue culture, and we report here that it does. EGF at a concentration of 25 ng/mL tissue culture medium lowers SH levels as compared with controls (0 ng/mL) in the epidermal layers examined (stratum malpighii of the tail and stratum malpighii and stratum corneum of flank skin). In general, other concentrations of EGF increase epidermal SH levels, although very high doses also reduce them. EGF at 25 ng/mL also lowers total SH + SS concentrations in the epidermal layers. Fetuses hemizygous for the Ta gene appear to have higher total SH + SS epidermal concentrations than their wild-type control littermates. These data, taken together with some of our previous findings, suggest the possibility that a relationship may exist between Ta, EGF, and thiol concentrations. Further study is required to elucidate this relationship.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Toluene Induction and Uptake Kinetics and Their Inclusion in the Specific-Affinity Relationship for Describing Rates of Hydrocarbon Metabolism

The kinetics of concentration-dependent toluene metabolism were examined by evaluating each term in the second-order rate equation. Marine and freshwater pseudomonads were used. Uptake for Pseudomonas sp. strain T2 was characterized by a completely saturatable system with small transport constant (K_t = 44 μg/liter) and large specific affinity. Kinetics for Pseudomonas putida PpF1 were similar. Induction had little effect on K_t, but it caused the specific affinity to increase from about 0.03 to 320 liters/g of cells per h. The level of induction depended on the time of exposure, the concentration of inducer, and the initial level of induction. If loss of the inducible system was not severe, toluene caused a linear increase in specific affinity with time, and the maximal value achieved at intermediate times (1 to 3 days) was hyperbolic with concentration when K_ind was 96 μg/liter (A. T. Law and D. K. Button, Appl. Environ. Microbiol. 51:469-476, 1986). As repression became complete, specific affinities were greatly reduced. Then induction required higher toluene concentrations and longer times, and the shape of the specific-affinity curve became sigmoidal with concentration. Cell yields (0.10 to 0.17 g of cells per g of toluene used) were low owing to liberation of organic products: 2-hydroxy-6-oxohepta-2,4-dienoic acid, toluene dihydrodiol, 3-methylcatechol, acetate, formate, and possibly pyruvate, which in turn caused lower rates of growth. Michaelis constants for the reaccumulation of products exceeded those for toluene, but specific affinities were lower and maximal velocities were higher, so that recycling was favored in cultures with high toluene concentration. Although these kinetics predict deviation from the linear relationship between uptake rate and biomass, we could detect none. Effects of saturation and induction were incorporated into the basic specific-affinity relationship. The result appears to be an improvement in the equation used for describing the kinetics of uptake and growth.     

Mentally abnormal offenders: manner of death.

A 23 year follow up of deaths in a population of mentally disordered patients was carried out, and a typical case history is reported. A quarter (71) of the deaths reported were unnatural, verdicts of suicide or accidental death or open verdicts having been recorded. For men in most age groups the proportion of deaths by suicide was two to three times greater than in the general population in 1982; the rate among those aged 25-29 was five times that in the general population. Differences in the rate of unnatural death among diagnostic categories of mental illness were not significant, but the proportion of unnatural deaths among the mentally handicapped will probably eventually be lower than that among psychotic offenders or those with personality disorders. Violent death occurs at an older age in those with affective disorders. Social isolation and alienation add to the handicap of mental disorder in this group of people, and these sometimes difficult but always vulnerable patients must continue to be offered asylum other than prison.     

The influence of crabs on litter processing in high intertidal mangrove forests in tropical Australia

Summary Measurements of litter fall and litter removal by crabs, in conjunction with estimates of litter decay by microbes and tidal export of litter from three high-intertidal mangrove forests were made during a year-long study in tropical northeastern Australia. In forests dominated by Ceriops tagal and Bruguiera exaristata, litter standing stocks remained low on the forest floor (mean 6 g·m^-2), although litter fall was high; 822 and 1022 g·m^-2·y^-1, respectively. Sesarmid crabs removed 580 (Ceriops) and 803 (Bruguiera) g·m^-2·y^-1, or 71 and 79%, of the total annual litter fall from the forest floor. Relative to the rate of litter removal by crabs, microbial turnover of whole, unshredded litter was insignificant, accounting for <1% of annual litter fall. Export of litter by tides was estimated to remove 194 (Ceriops) and 252 (Bruguiera) g·m^-2·y^-1 or 24 and 25% of annual litter fall. In a forest dominated by Avicenniamarina, in which an ocypodid crab was more abundant than sesarmids, litter standing stocks were higher (mean 84 g·m^-2) and crabs removed less litter; 173 g·m^-2·y^-1 or 33% of the annual litter fall of 519 g·m^-2·y^-1. Microbial turnover of intact litter was more important in the Avicennia forest (168 g·m^-2·y^-1 or 32% of annual litter fall), and tides exported 107 g·m^-2·y^-1 or 21% of litter production. In areas where sesarmid crabs were absent or rare in Ceriops forests, there were significantly higher standing stocks of litter and slower rates of leaf removal. Taking into account the probable assimilation efficiencies of sesarmid crabs feeding on mangrove leaves, we estimate that in Ceriops and Bruguiera forests leaf processing by crabs turns litter over at >75 times the rate of microbial decay alone, thus facilitating the high sediment bacterial productivity in these forests. The importance of litter processing by crabs increases with height in the intertidal in tropical Australia, in contrast to New World mangrove forests, where the reverse is true.Contribution No. 445 from the Australian Institute of Marine Science     

Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.     

Binding of the 3'' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.     

Modified P Elements That Mimic the P Cytotype in Drosophila Melanogaster

Activity of the P family of transposable elements in Drosophila melanogaster is regulated primarily by a cellular condition known as P cytotype. It has been hypothesized that P cytotype depends on a P element-encoded repressor of transposition and excision. We provide evidence in support of this idea by showing that two modified P elements, each with lesions affecting the fourth transposase exon, mimic most of the P cytotype effects. These elements were identified by means of two sensitive assays capable of detecting repression by a single P element. One assay makes use of cytotype-dependent gene expression of certain P element insertion mutations at the singed bristle locus. The other measures suppression of transposase activity from the unusually stable genomic P element, delta 2-3(99B), that normally produces transposase in both germinal and somatic tissues. The P cytotype-like effects include suppression of snw germline hypermutability, snw somatic mosaicism, pupal lethality, and gonadal dysgenic sterility. Unlike P cytotype, however, there was no reciprocal cross effect in the inheritance of repression.     

High-frequency disruption of the N-myc gene in embryonic stem and pre-B cell lines by homologous recombination.

Identification of gene function has often relied on isolation of mutant cells in which expression of the gene was inactivated. Gene targeting by homologous recombination in tissue culture now may provide a technology to rapidly and directly produce such mutant mammalian cells. We demonstrate that selection of embryonic stem and pre-B cell lines for expression of a promoterless construct containing murine N-myc genomic sequences fused to a gene encoding neomycin resistance allows highly efficient recovery of variants in which the endogenous N-myc gene is disrupted. The high frequency of N-myc gene disruption by this method should permit targeted disruption of both allelic N-myc copies in various cell lines to study N-myc function.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.