Metabolism As a Function of Water Potential in Air-Dry Seeds of Charlock (Sinapis arvensis L.)

A new method is described for studying the metabolism of air-dry seeds. An initial pulse of (14)CO(2) was supplied to seeds maintained in air at controlled low water potentials for 6 months. Seeds were also infiltrated with 2-(14)C-acetate and with (14)C-l-leucine at 0 C, redried rapidly at 0 C, and maintained at controlled low water potentials for 4 to 6 weeks. The metabolism of the air-dry seeds was a function of the water content of the tissues, which was in equilibrium with the water potential at the seed surface. The fixation of (14)CO(2) and the utilization of 2-(14)C-acetate increased exponentially with water content. The incorporation of (14)C-l-leucine into protein increased linearly with water content. Metabolism was not reduced to a low rate except in air-dry seeds at the lowest water potentials (-1716 to -762 bars) with 4 to 6% water.     

Comparison of effects of prolactin and growth hormone on adrenal 5alpha-reductase in hypophysectomized rats.

Adrenal 5alpha-reductase activity was measured in female rats 0, 2, 5, and 6 days after hypophysectomy. Enzyme activity increased progressively exhibing a 35-fold elevation at 6 days. The effects of high (250 mug/100 g of body wt), intermediate (25 mug/100 g of body wt), and low (2.5 mug/100 of body wt) daily doses of bovine prolactin and bovine growth hormone were compared at 2 and 5 days posthypophysectomy. At 2 days, enzyme activity was partially inhibited by the high and intermediate doses of prolactin and not affected by growth hormone. At 5 days all doses of prolactin were inhibitory, whereas enzyme activity was suppressed only by the high dose of growth hormone. With a given dose of hormone, the amount of suppression of enzyme activity is greater at 5 days than at 2 days posthypophysectomy. In 5-day hypophysectomized rats the inhibitory effects of prolactin and growth hormone were additive. It is concluded that: (i) hormonal sensitivity and responsiveness of the adrenal reductase pathway increases with duration of pituitary ablation; (ii) the reductive pathway is more sensitive to the effects of prolactin than growth hormone; and (iii) the effects of growth hormone and prolactin on reductase activity are mediated via different mechanisms, as suggested by the additive effects of individual hormones.     

Purification and properties of an esterase B4 from human liver.

A butyryl esterase, designated B4, has been purified from human liver and some of its properties described. The activity of this enzyme comprises 0.48% of the total butyryl esterase activity found in human liver. Esterase B4 has been distinguished from other butyryl esterases by its preference for the esters of the fluorogenic compounds 4-methyl umbilliferone and fluorescein over naphthyl esters as substrates. Other distinguishing features of this esterase include a relatively high pI (pH 8.7) A monomeric structure of low molecular weight (20 000) and high solubility in solutions of ammonium sulphate.     

Screening methods for covert bacteriuria in schoolgirls.

Screening tests for bacteriuria based on two different principles were evaluated in1582 schoolgirls aged 5-11 years, and in 26 girls aged 3-16 years attending hospitalwith symptomatic urinary tract infection. Tests for hypoglucosuria, performed by a semi-automated fluorometric method and with Uriglox strips on early-morning urine samples voided after overnight fasting, gave unacceptably high false-negative rates (16.7% and 20.8% respectively). Oxoid and Uricult dipslides were immersed in fresh midstreamspecimens of urine obtained at school and read overnight incubation at 37 degrees C.Both gave comparable results, with low false-positive rates and no false-negative responses. The higher cost of screening by dipslides was halved by using the "dipstream" technique, which also gave no false-negative results. Its false-positive rate of 13.5% could be reduced to 1.8% by disregarding colony counts of 10-8 non-faecal organisms and over per litre, which appear unimportant in schoolchildren. Bacteriuria was found in 2.3% of the schoolgirls; 39% of them had symptons, compared with 7.2% of the healthy girls, and 25% showed vesicoureteric reflux, which in 17% was associated with renalscarring. Since the natural history of covert bacteriuria and its relationship withreflux and scarring remain undetermined further research is required. The dipstreamtechnique offers a simple, reliable, and comparatively cheap screening method which could also be applied in general practice.     

An abnormality of oestrogen feedback in amenorrhoea-galactorrhoea.

Fourteen patients with amenorrhoea and hyperprolactin-anemia but no evidence of pituitary tumours were each given an intramuscular injection of 1 mg oestradiol benzoate. Thirteen patients failed to release luteinizing hormone in response to the oestrogen. This hypothalamic abnormality may help to explain the menstrual disturbances in subjects with hyperprolactinaemia.     

Construction and characterisation of a yeast artificial chromosome library containing five haploid sugarbeet (Beta vulgaris L.) genome equivalents

A yeast artificial chromosome (YAC) genomic library of Beta vulgaris was constructed in the pYAC4 vector. High-molecular-weight DNA was prepared from agarose-embedded leaf protoplasts from a triploid cultivar. The library was found to contain 33,500 clones in an ordered array of microtiter plates. Mean size of the inserts was estimated to be 135 kb, and the library should therefore represent the equivalent of five haploid genomes. The library was characterised for the presence of highly repetitive, chloroplast and single-copy sequences. In order to isolate single-copy sequences, 18 pools of DNA, each from 1920 individual YAC clones, were prepared for rapid screening of the library by the polymerase chain reaction. The results of these screenings showed that the number of isolated clones was at or near the frequency expected.     

Rapid assessment of bacterial viability by flow cytometry

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC₆(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDAthan with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.Correspondence to: J. P. Diaper     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

Respiratory chain composition and activity in some thermotolerant streptomycetes

The cytochrome composition of membrane preparations from 3 thermotolerant species of Streptomyces was determined from difference spectra. All contained b, c and a-type haemoproteins and in addition, 2 species (Streptomyces thermoviolaceus and Streptomyces thermoflavus) possessed cytochrome oxidase d. A CO-reacting b cytochrome similar to cytochrome oxidase o could also be detected in all 3 organisms. Thermotolerance of the respiratory chains showed no correlation with cytochrome composition. In general, Streptomyces thermonitrificans had the most thermostable activities, whilst those of S. thermoviolaceus were the most thermolabile. Growth temperature exerted no qualitative effects on respiratory chain composition but did affect the thermostability of some substrate-supported oxidase activities.