Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide

Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr left arrow over right arrow Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.     

A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 micrograms; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.     

Radiation-induced enhanced proliferation of human squamous cancer cells in vitro: a release from inhibition by epidermal growth factor

Ionizing radiation is believed to stimulate the repopulation of squamous carcinoma cells that survive the early portion of a fractionated course of radiotherapy. To characterize any intrinsic radiation-induced adaptive response and to examine whether epidermal growth factor (EGF) influences this response, A431 and 183A cells were irradiated with repeated daily exposures of 0.5-0.75 Gy and then grown in monolayer culture for 7 days with or without EGF at a 1 ng/ml concentration. Cell numbers were quantified using a microtiter dye-reduction assay. EGF alone caused approximately 70% and 30% growth inhibition of human SC A431 and 183A cells, respectively. Although radiation alone did not affect proliferative rates in these conditions, radiation eliminated the EGF-related growth inhibition in both cell lines. This effect was dose dependent in single radiation exposure experiments. Cell cycle analyses indicated that EGF initially promoted entry into S-phase 3 days after treatment but caused a G1-S block after 7 days. Treatment with radiation recruited cells into S-phase and G2-M, an effect which was sustained 7 days after treatment, overriding the influence of EGF. Radiation-induced modulation of the response of human squamous carcinoma cells to EGF in vitro after single and repeated radiation exposures suggests a proliferation response that may underlie enhanced repopulation of tumor clonogens in vivo.     

Computational learning reveals coiled coil-like motifs in histidine kinase linker domains

The recent rapid growth of protein sequence databases is outpacing the capacity of researchers to biochemically and structurally characterize new proteins. Accordingly, new methods for recognition of motifs and homologies in protein primary sequences may be useful in determining how these proteins might function. We have applied such a method, an iterative learning algorithm, to analyze possible coiled coil domains in histidine kinase receptors. The potential coiled coils have not yet been structurally characterized in any histidine kinase, and they appear outside previously noted kinase homology regions. The learning algorithm uses a combination of established sequence patterns in known coiled coil proteins and histidine kinase sequence data to learn to recognize efficiently this coiled coil-like motif in the histidine kinases. The common appearance of the structural motif in a functionally important part of the receptors suggests hypotheses for kinase regulation and signal transduction.     

Screening calcaneal ultrasound and risk factors for osteoporosis among lesbians and heterosexual women

We compared calcaneal ultrasound measurements (speed of sound [SOS], broadband ultrasound attenuation [BUA], and stiffness index [SI]) of lesbian and heterosexual women to examine the medical and lifestyle risk factors for osteoporosis in each group. This was an exploratory, community-based, cross-sectional study. Subjects were mailed food frequency, health, and physical activity questionnaires. Weight, height, and calcaneal ultrasound measurements were taken at one office visit. In communities in southern and eastern Maine, 71 lesbians and 77 heterosexual women between the ages of 30 and 50 with regular menses and in good general health were the subjects. Statistical analysis used t-tests and chi-square tests to evaluate differences between study groups. Linear regression models were used to evaluate risk factors for low calcaneal ultrasound measurements. There were no differences between the lesbian and heterosexual women in age, body mass index (BMI), exercise, calcium intake, alcohol use, or calcaneal ultrasound measurements. There was a positive association between BUA and both BMI and alcohol consumption (p < 0.01). Antidepressant use significantly reduced SOS and SI (p < 0.05). There were no differences in calcaneal ultrasound measurements between lesbian and heterosexual women. BMI was strongly and positively associated with BUA. Antidepressant use in both populations was associated with a significant reduction in calcaneal bone mass. Studies are needed to define the relationship of depression and its treatment to bone mineral density and the future risk of osteoporosis.     

The athlete's heart: is this an entity in paediatrics?

A case report of a boy who demonstrates features of an athlete's heart, associated with dilatation of the coronary artery, is presented. The importance of distinguishing this benign condition from pathologic causes such as cardiomyopathy, and risk of sudden death in these athletes is discussed.     

Increased transversions in a novel mutator colon cancer cell line

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.