Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase

Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.     

Variation in the skull of the Long-tailed field-mouse, Apodemus sylvaticus in mainland Britain

Nine skull measurements and a measure of age have been made on each of 613 specimens of Apodemus sylvaticus (L.). The mice were collected from 12 mainland and three island localities. From five of the localities they were obtained in more than one year; each year's collection was kept separate in the subsequent analysis.
Unadjusted and adjusted (for age) means have been calculated for each character. Differences in both space and time are small for the mainland populations. A discriminant function analysis was undertaken with a view to accounting for the larger part of the variation using a limited number of linear combinations of the adjusted measurements. It was found that the greater part of the variation was contained in the first two canonical variates. Finally, the generalized distances were obtained.
The multivariate analyses suggest that one island population (St Mary's) is quite distinct, the other two (Tresco, Mull) less so, that there is in any one locality little variation from year to year, that the Scottish mainland populations as a group display a small statistical divergence from the English mainland populations and that the former differ rather more from each other than do the latter.     

Mute Swans in Great Britain: a review,current status and long-term trends

We review the recent history of the Mute Swan in Great Britain and discuss the factors known to be affecting the population. Following a rise in the national population during the 1950s, numbers decreased sharply during the 1960s, and changed relatively little between 1970/71 and 1984/85. However, there has been considerable regional variation in the fortunes of Mute Swan populations during this period, with dramatic declines in some areas. Although several factors were thought to be contributing to such declines, poisoning from the ingestion of lead fishing weights was shown to be the largest single cause of death amongst swans in a number of areas. Voluntary measures to address this problem were initiated in 1982 and culminated in the banning by law of use of lead weights in 1987.Winter counts were used to investigate the current status and distribution of the Mute Swan in Great Britain and to examine long-term regional trends. The maximum total count reached 12600 birds in January 1990, which compares with an average of 9550 for the previous five winters. However, accounting for birds missed, the population may now number at least 25 000. Peak total numbers have mostly occurred in September, after which numbers remain approximately stable until December and then decline. Patterns of seasonal abundance vary between regions and habitats and these are discussed.The British population has increased dramatically since 1986/87 and reached its highest level for 27 years in 1987/88. There have been recent increases in most regions with record levels being reached mostly in 1987 or 1988, and there has been growth in the numbers on all habitat types, especially on reservoirs, gravel extraction pits and freshwater marshes. The timing of these increases corresponds very closely with the introduction of legislation against the use of lead fishing weights, and the incidence of lead poisoning is known to have been considerably reduced by such measures.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Mapping of the chicken cleft primary palate mutation on chromosome 11 and sequencing of the 4.9 Mb linked region

An embryonic lethal mutation in chicken named cleft primary palate (cpp) is inherited in an autosomal recessive mode and results in a severely truncated upper beak. In this study, genotyping and sequencing techniques were employed to advance our genetic and genomic knowledge of the mutation’s chromosomal location, candidate region and possible causative element using a congenic inbred line. Herein, the candidate region for the cpp developmental mutation was established as a ca. 5.1 Mb region of chicken chromosome 11 (GGA 11) through the use of a 600K Affymetrix SNP array. The SNPs identified from this array linked to cpp were used to genotype individuals from the congenic inbred line over several generations and thereby fine-map the causative region resulting in an approximately 200 kb size reduction. This candidate region (4.9 Mb) was sequenced via capture array in a cohort of 24 individuals, including carriers, mutants and their wild type (wt) siblings. Interestingly, the GGA 11 region for cpp encompasses the predicted centromere location and is thus unlikely to be highly disrupted by further recombination. Here we report on the variation unique to the cpp mutation, i.e. single-nucleotide variants and insertions or deletions. Although the candidate region contains several genes of interest with regard to the cpp phenotype, only one cpp-linked variant was predicted to have a significant physiological effect by causing a frameshift mutation in ESRP2, which has a role in tissue-specific splicing during development.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.