Biochemistry,genetics and physiology of microbial styrene degradation

The last few decades have seen a steady increase in the global production and utilisation of the alkenylbenzene, styrene. The compound is of major importance in the petrochemical and polymer-processing industries, which can contribute to the pollution of natural resources via the release of styrene-contaminated effluents and off-gases. This is a cause for some concern as human over-exposure to styrene, and/or its early catabolic intermediates, can have a range of destructive health effects. These features have prompted researchers to investigate routes of styrene degradation in microorganisms, given the potential application of these organisms in bioremediation/biodegradation strategies. This review aims to examine the recent advances which have been made in elucidating the underlying biochemistry, genetics and physiology of microbial styrene catabolism, identifying areas of interest for the future and highlighting the potential industrial importance of individual catabolic pathway enzymes.     

Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway

The concentration of glucose in plasma is an important determinant of pancreatic beta-cell mass, whereas the relative contributions of hypertrophy, proliferation, and cell survival to this process are unclear. Glucose results in depolarization and subsequent calcium influx into islet beta-cells. Because depolarization and calcium (Ca(2+)) influx promote survival of neuronal cells, we hypothesized that glucose might alter survival of islet beta-cells through a similar mechanism. In the present studies, cultured mouse islet beta-cells showed a threefold decrease in apoptosis under conditions of 15 mM glucose compared with 2 mM glucose (P < 0.05). MIN6 insulinoma cells incubated in 25 mM glucose for 24 h showed a threefold decrease in apoptosis compared with cells in 5 mM glucose (1.7 +/- 0.2 vs. 6.3 +/- 1%, respectively, P < 0.001). High glucose (25 mM) enhanced survival-required depolarization and Ca(2+) influx and was blocked by phosphatidylinositol (PI) 3-kinase inhibitors. Glucose activation of the protein kinase Akt was demonstrated in both insulinoma cells and cultured mouse islets by means of an antibody specific for Ser(473) phospho-Akt and by an in vitro Akt kinase assay. Akt phosphorylation was dependent on PI 3-kinase but not on MAPK. Transfection of insulinoma cells with an Akt kinase-dead plasmid (Akt-K179M) resulted in loss of glucose-mediated protection, whereas transfection with a constitutively active Akt enhanced survival in glucose-deprived insulinoma cells. The results of these studies defined a novel pathway for glucose-mediated activation of a PI 3-kinase/Akt survival-signaling pathway in islet beta-cells. This pathway may provide important targets for therapeutic intervention.     

Rectal and colonic distension elicit viscerovisceral reflexes in humans

Colonic transit is slowed in patients with disordered rectal evacuation, but the mechanism of this phenomenon is unclear. Our objective was to investigate rectocolonic inhibitory reflexes in humans to provide potential insight into patients with obstructed defecation. In 30 healthy subjects, a barostat-manometric assembly recorded colonic tone and phasic activity in the descending colon during rectal distension and recorded rectal tone during colonic distension. Phasic distensions were 8, 16, and 32 mmHg above balloon operating pressure, and staircase inflations were comprised of balloon inflation then deflation in 2-mmHg increments at 30-s intervals from 0 to 36 mmHg. Colonic balloon volumes increased to a similar extent during phasic rectal distensions 8, 16, and 32 mmHg above operating pressure, reflecting reduced colonic tone; balloon volumes also increased and phasic pressure activity decreased during staircase rectal distensions. In contrast, rectal balloon volume declined, reflecting increased tone during phasic and staircase colonic distensions. Thus rectal distension inhibited colonic motor activity, indicative of a viscerovisceral inhibitory reflex.     

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.     

ELAC2 and prostate cancer risk in Afro-Caribbeans of Tobago

To test the hypothesis that variation in the putative prostate cancer susceptibility gene ELAC2 contributes to the elevated risk of prostate cancer in Afro-Caribbean males from Tobago, we genotyped the S217L and A514T polymorphisms, previously reported to be associated with prostate cancer risk in a large sample of cases and controls. The frequency of the high-risk Leu allele at the S217L site was the same in cases and controls. Both cases and controls were homozygous for the low-risk Ala allele at the A514T site. In addition, we sequenced the exons and 3'- and 5'-flanking regions of ELAC2 in 24 individuals with histologically confirmed prostate cancer. We identified 17 new single nucleotide polymorphisms. An A(-1196)T polymorphism, which alters a predicted TATA box consensus sequence, was tested in cases and controls, and no significant difference in allele or genotype frequencies was observed. The absence of ELAC2 mutations and lack of association between polymorphisms in ELAC2 and prostate cancer in cases and controls leads us to conclude that ELAC2 does not contribute significantly to the elevated prevalence of prostate cancer in Afro-Caribbean males of Tobago.     

Identifying and managing adverse environmental health effects: 2. Outdoor air pollution

AIR POLLUTION CONTRIBUTES TO PREVENTABLE ILLNESS AND DEATH. Subgroups of patients who appear to be more sensitive to the effects of air pollution include young children, the elderly and people with existing chronic cardiac and respiratory disease such as chronic obstructive pulmonary disease and asthma. It is unclear whether air pollution contributes to the development of asthma, but it does trigger asthma episodes. Physicians are in a position to identify patients at particular risk of health effects from air pollution exposure and to suggest timely and appropriate actions that these patients can take to protect themselves. A simple tool that uses the CH²OPD² mnemonic (Community, Home, Hobbies, Occupation, Personal habits, Diet and Drugs) can help physicians take patients'' environmental exposure histories to assess those who may be at risk. As public health advocates, physicians contribute to the primary prevention of illness and death related to air pollution in the population. In this article we review the origins of air pollutants, the pathophysiology of health effects, the burden of illness and the clinical implications of smog exposure using the illustrative case of an adolescent patient with asthma.Case A 16-year-old girl and her mother visit their family physician in July because the daughter woke up at 6 am that morning with shortness of breath, a cough and tightness in her chest. The girl has a history of asthma and used salbutamol soon after the onset of symptoms, with some but not total relief. She reports having had no symptoms during the previous month. She had a few episodes of wheezing the previous summer, which resolved with the use of salbutamol, and a cough that persisted for 2 weeks after an upper respiratory tract infection in the winter. She has no history of allergies, hayfever or other medical problems. She is a nonsmoker and has no family history of allergies. Audible wheezing is detected on physical examination, but the girl does not appear to be in distress. Her vital signs are normal, as are the results of the ear-nose-throat and cardiovascular examinations. Respiratory examination reveals wheezing throughout chest, no focal findings and a centrally placed trachea. The girl''s calves are soft and nontender, and there is no evidence of ankle edema. Her peak expiratory flow is 240 L/min (expected for height 400 L/min). Spirometry testing is unavailable. Fifteen minutes after 2 puffs of salbutamol her peak expiratory flow increases to 320 L/min. To identify possible exposures that may have contributed to the asthma episode, the physician quickly takes an environmental exposure history using the CH²OPD² mnemonic — Community, Home, Hobbies, Occupation, Personal habits, Drugs and Diet (1Table 1Open in a separate windowQuestions surrounding this case: What was the patient''s exposure to outdoor air pollutants? How should the patient and family be counselled about dealing with these trigger factors? What are the possible inducers and triggers from indoor air pollution? How can the patient and family find out about the status of outdoor air quality in their community?     

Signal transduction in the photoactive yellow protein. II. Proton transfer initiates conformational changes

Molecular dynamics simulation techniques, together with semiempirical PM3 calculations, have been used to investigate the effect of photoisomerization of the 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. In this bacteria, exposure to blue light leads to a negative photoactic response. The calculations suggest that the isomerization does not directly destabilize the protein. However, because of the isomerization, a proton transfer from a glutamic acid residue (Glu46) to the phenolate oxygen atom of the chromophore becomes energetically favorable. The proton transfer initiates conformational changes within the protein, which are in turn believed to lead to signaling.     

Structural determinants of aromatase cytochrome p450 inhibition in substrate recognition site-1

The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I(133)M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I(133)M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile(133) to either alanine (I(133)A) or tyrosine (I(133)Y) decreased apparent enzyme activity, but the I(133)A mutant was sensitive to etomidate inhibition, suggesting that it is Ile(133) that decreases etomidate binding rather than Met(133) increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I(133)M mutant and the native placental isozyme. These data suggest that Ile(133) is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.     

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.