Butanol production in S. cerevisiae via a synthetic ABE pathway is enhanced by specific metabolic engineering and butanol resistance.

BACKGROUND: The fermentation of sugars to alcohols by microbial systems underpins many biofuel initiatives. Short chain alcohols, like n-butanol, isobutanol and isopropanol, offer significant advantages over ethanol in terms of fuel attributes. However, production of ethanol from resistant Saccharomyces cerevisiae strains is significantly less complicated than for these alternative alcohols. RESULTS: In this study, we have transplanted an n-butanol synthesis pathway largely from Clostridial sp. to the genome of an S. cerevisiae strain. Production of n-butanol is only observed when additional genetic manipulations are made to restore any redox imbalance and to drive acetyl-CoA production. We have used this butanol production strain to address a key question regarding the sensitivity of cells to short chain alcohols. In the past, we have defined specific point mutations in the translation initiation factor eIF2B based upon phenotypic resistance/sensitivity to high concentrations of exogenously added n-butanol. Here, we show that even during endogenous butanol production, a butanol resistant strain generates more butanol than a butanol sensitive strain. CONCLUSION: These studies demonstrate that appreciable levels of n-butanol can be achieved in S. cerevisiae but that significant metabolic manipulation is required outside of the pathway converting acetyl-CoA to butanol. Furthermore, this work shows that the regulation of protein synthesis by short chain alcohols in yeast is a critical consideration if higher yields of these alcohols are to be attained.

Beyond power laws: a new approach for analyzing single molecule photoluminescence intermittency.

The photoluminescence intermittency (PI) exhibited by single emitters has been studied for over a decade. To date, the vast majority of PI analyses involve parsing the data into emissive and non-emissive events, constructing histograms of event durations, and fitting these histograms to either exponential or power law probability distributions functions (PDFs). Here, a new method for analyzing PI data is presented where the data are used directly to construct a cumulative distribution function (CDF), and maximum-likelihood estimation techniques are used to determine the best fit of a model PDF to the CDF. Statistical tests are then employed to quantitatively evaluate the hypothesis that the CDF (data) is represented by the model PDF. The analysis method is outlined and applied to PI exhibited by single CdSe∕CdS core-shell nanocrystals and the organic chromophore violamine R isolated in single crystals of potassium-acid phthalate. Contrary to previous studies, the analysis presented here demonstrates that the PI exhibited by these systems is not described by a power law. The analysis developed here is also used to quantify heterogeneity within PI data obtained from a collection of CdSe/CdS nanocrytals, and for the determination of statistically significant changes in PI accompanying perturbation of the emitter. In summary, the analysis methodology presented here provides a more statistically robust approach for analyzing PI data.

Tobacco spending and children in low income households.

OBJECTIVE: To examine the role of tobacco use in creating financial hardship for New Zealand (NZ) low income households with children. DATA: The 1996 NZ census (smoking prevalence by household types), Statistics NZ (household spending surveys 1988-98), and NZ Customs (tobacco released from bond 1988-98). MAIN OUTCOME MEASURES: Proportion of children in households with smokers and < or = 15,000 NZ dollars gross income per adult. Proportion of spending on tobacco of second lowest equivalised household disposable income decile and of solo parent households. RESULTS: In < or = 15,000 NZ dollars gross income per adult households with both children and smokers, there were over 90,000 children, or 11% of the total population aged less than 15 years. Enabling second lowest income decile households with smokers to be smoker-free would on average allow an estimated 14% of the non-housing budgets of those households to be reallocated. CONCLUSIONS: The children in low income households with smokers need to be protected from the financial hardship caused by tobacco use. This protection could take the form of more comprehensive government support for such households and stronger tobacco control programmes. A reliance on tobacco price policy alone to deter smokers is likely to have mixed outcomes-for example, increased hardship among some of these households. The challenge for tobacco control is to move from a sole focus on "doing good" towards incorporating the principle of "doing no harm".

Time-resolved phosphorescence of tyrosine, tyrosine analogs, and tyrosyl residues in oxytocin and small peptides.

We present the time-resolved phosphorescence of oxytocin, two oxytocin derivatives, vasopressin and a series of compounds that serve as models for free tyrosine. One of the oxytocin derivatives, desaminodicarbaoxytocin, has the disulfide bridge replaced by an ethylene bridge, and lacks the N-terminus. Similar to the reported fluorescence decays of tyrosine in these peptides, the phosphorescence decays generally are not single exponentials, but can be fit as biexponentials. The decay times for the oxytocin peptides are shorter than for desaminodicarbaoxytocin or the model compounds, and this we attribute to enhanced spin-orbit coupling due to the presence of sulfur. We measured the phosphorescence decay of the model cyclic pentapeptide that contains tyrosine and compared it to that observed for the same cyclic pentapeptide in which tyrosine is replaced by tryptophan. We also report the phosphorescence of 2-tryptophan-oxytocin, and deamino-2-tryptophan-oxytocin in which biexponential phosphorescence decay is also observed.

Understanding the phase-dependent reactivity of chlorine dioxide using resonance Raman spectroscopy.

Progress in understanding the phase-dependent reactivity of chlorine dioxide (OClO) is outlined. Resonance Raman intensity analysis studies of gaseous and solution-phase OClO are presented which demonstrate that the optically prepared excited state undergoes significant modification in solution. In addition, time-resolved resonance Raman studies are presented which demonstrate that geminate recombination of the primary photoproducts, resulting in the re-formation of ground-state OClO, dominates the photochemical reaction dynamics in solution. The current picture of aqueous OClO photochemistry derived from these studies is discussed, and future directions of investigation are outlined.

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo.

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway.

FTIR studies of intermolecular hydrogen bonding in halogenated ethanols.

The effect of halogen substitution on intermolecular hydrogen-bonding in ethanol is studied. Specifically, Fourier-transform infrared (FTIR) spectra of ethanol, 2,2,2-trifluoroethanol (TFE), and 2,2,2-trichloroethanol dissolved in carbon tetrachloride are reported as a function of temperature and concentration. The spectral intensities corresponding to monomer, dimer, and multimer formation are used to determine the effect of halogen substitution on intermolecular hydrogen-bonding. The enthalpy for dimerization was found to evolve from -4.2+/-0.3 kcal/mol in ethanol to -6.8+/-1.0 kcal/mol in TFE. An opposite trend was observed for multimer formation with enthalpies of -3.7+/-0.5 in ethanol and -2.1+/-1.4 kcal/mol in TFE. The majority of this evolution is assigned to the ability of ethanols to form intramolecular hydrogen bonds involving the hydoxyl proton and the halogen substituents.

Disruption and functional analysis of six ORFs on chromosome XII of saccharomyces cerevisiae: YLR124w, YLR125w, YLR126c, YLR127c, YLR128w and YLR129w.

In the context of the EUROFAN programme, we report the deletion and functional analysis of six open reading frames (ORFs) on the right arm of chromosome XII of Saccharomyces cerevisiae. Using a PCR-based gene replacement strategy, we have systematically deleted individual ORFs and subjected the heterozygous diploids and haploid knockout strains to basic genetic and phenotypic characterization. Two ORFs, YLR127c and YLR129w, are essential for viability, whereas no growth phenotype could be detected following deletion of YLR124w, YLR125w, YLR126c or YLR128w. For each of the individual ORFs, a kanMX4 replacement cassette and the corresponding cognate wild-type gene were cloned into appropriate plasmids.

Distinct domains within yeast Sec61p involved in post-translational translocation and protein dislocation.

The translocation of secretory polypeptides into and across the membrane of the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. Recent data also suggest that misfolded or unassembled polypeptides exit the ER via the translocon for degradation by the cytosolic ubiquitin/proteasome pathway. Sec61p is a highly conserved multispanning membrane protein that constitutes a core component of the translocon. We have found that the essential function of the Saccharomyces cerevisiae Sec61p is retained upon deletion of either of two internal regions that include transmembrane domains 2 and 3, respectively. However, a deletion mutation encompassing both of these domains was found to be nonfunctional. Characterization of yeast mutants expressing the viable deletion alleles of Sec61p has revealed defects in post-translational translocation. In addition, the transmembrane domain 3 deletion mutant is induced for the unfolded protein response and is defective in the dislocation of a misfolded ER protein. These data demonstrate that the various activities of Sec61p can be functionally dissected. In particular, the transmembrane domain 2 region plays a role in post-translational translocation that is required neither for cotranslational translocation nor for protein dislocation.

Detection of massive transplacental haemorrhage by flow cytometry.

Flow cytometry has been shown to be a more accurate and sensitive method than the Kleihauer-Betke test for the measurement of feto-maternal haemorrhage in Rh(D) incompatibility. This report describes the successful use of flow cytometry to detect and monitor the management of a massive transplacental haemorrhage (105 ml) of fetal Rh(D) positive cells in a Rh(D) negative woman. The report highlights the accuracy and reproducibility of the test and the stability of a blood sample when transferred 596 kilometres to a central testing facility.

Mortality of white South African gold miners.

OBJECTIVES: This two part study aimed to determine whether there was an excess mortality generally or for some diseases among middle aged white South African gold miners on the Witwatersrand and whether the underground dust exposure of these miners contributed to the development of lung cancer, chronic obstructive pulmonary disease (COPD), or ischaemic heart disease (IHD). METHODS: A cohort of 4925 white miners in South Africa, born between 1 January 1916 and 31 December 1930 who were alive and working in the vicinity of Johannesburg on 1 January 1970, then aged between 39 and 54, was followed up for 20 years by which time 2032 had died. Most were gold miners (about 87% had worked 85% or more of their shifts in gold mines). Standardised mortality ratios (SMRs) were calculated as percentages of the number of deaths observed in the cohort for a condition as stated on the death certificate divided by the number expected on the basis of concurrent mortality in the reference population (the total age specific white male population of South Africa). A case-control analysis was performed for three diseases (lung cancer, COPD, and IHD), the results of which are presented for those miners in the cohort who had spent at least 85% of their service on gold mines and had worked at least 15% of their shifts underground. RESULTS: The SMR for all causes of death was 129.6%, raised because of excess mortality due to the following causes: lung cancer (SMR = 139.8%), IHD (124.1%), COPD (189%) and cirrhosis of the liver (155.3%). Smoking was confirmed to be the main risk factor for lung cancer and COPD although cumulative dust exposure was found to increase the risk of COPD in conjunction with smoking. No significant risk of lung cancer resulted from exposure to dust. High blood pressure and smoking were found to increase the risk of IHD, but no association between IHD and the quetelet index (weight/height2) was found. CONCLUSIONS: The most significant and unexpected finding was the 30% increase in the SMR for all causes. Very little of this increase could be attributed to mining and the main factor for this was probably the adoption of an unhealthy lifestyle by these men (compared with other South African white men) particularly smoking (86% were smokers) and alcohol consumption.

Detection of cryptic prey: search image or search rate?

Animals' improvement in capturing cryptic prey with experience has long been attributed to a perceptual mechanism, the specific search image. Detection could also be improved by adjusting rate of search. In a series of studies using both naturalistic and operant search tasks, pigeons searched for wheat, dyed to produce 1 conspicuous and 2 equally cryptic prey types. Contrary to the predictions of the search-rate hypothesis, pigeons given a choice between the 2 cryptic types took the type experienced most recently. However, experience with 1 cryptic type improved accuracy on the other cryptic type, a result inconsistent with a search image specific to 1 prey type. Search image may better be thought of as priming of attention to those features of the prey type that best distinguish the prey from the background.

Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine.

The metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine (3TC) was examined in human immunodeficiency virus type 1 (HIV-1)-infected and mock-infected human cells. 3TC 5'-triphosphate levels accumulated comparably in HIV-1-infected and mock-infected phytohaemagglutinin-stimulated peripheral blood lymphocytes (PBL) and reached 40% or more of total intracellular 3TC metabolites after 4 hr. The rate of decay of 3TC triphosphate in HIV-1-infected and mock-infected PBL measured as a half-life (T1/2) ranged from 10.5 to 15.5 hr. 3TC did not significantly affect metabolism of deoxynucleotides in the U937 cell line, and was shown to be resistant to the action of human platelet pyrimidine nucleoside phosphorylase.

Replicated testing of the Nursing Technology Model.

The purpose of this study was to replicate the testing of a model to explain complexity of nursing care in the ambulatory care setting. The Nursing Technology Model (Verran & Shaw, 1986) has as its theoretical base a sociological perspective of organizational analysis in which technology is viewed as the antecedent to organizational structure. The model includes materials technology as the nature of the ambulatory care client and knowledge technology as the principal type of activities pertinent to nursing care delivery. The characteristics of these technology types were organized into causal paths to explain complexity of care. Two ambulatory care sites were sampled to obtain a client sample of 610 rating sets. Results were essentially similar to earlier research with R2s for complexity indexes at the same level of .34. Only one regression equation evidenced instability in regression coefficients and the R2. Analysis of findings indicated that other variables, such as some aspects of structure, may need to be further specified in the model in order to increase its explanatory power.

Automatic implantable cardioverter-defibrillator: appearance and complications.

The automatic implantable cardioverter-defibrillator (AICD) can revert life-threatening arrhythmias to normal rhythms in ambulatory patients. The device continually monitors cardiac rhythm, and delivers cardioverting discharges when potentially life-threatening arrhythmias are recognized. The radiographs of 22 patients were reviewed with special reference to complications of radiologic interest, including pneumothorax, infiltrate, pleural effusion, atelectasis, and malposition of sensing or defibrillating leads. The normal radiographic appearance, surgical implantation, and clinical aspects of the AICD are discussed. A potential pitfall, an apparent connection gap at the epicardial electrode, was seen in three patients. This is due to limited radiodensity of part of the conduit. The AICD can lead to substantial improvement in survival rates of properly selected patients. Increasing use is probable, and radiologists should be aware of the normal appearance and complications of the AICD.

The molecular basis of suppression in an ochre suppressor strain possessing altered ribosomes.

Escherichia coli K12 2320(lambda)-15B has a mutation that results in ochre suppressor activity.(1) This mutation concomitantly causes a decreased growth rate in rich medium, an increased sensitivity to streptomycin,(1) and the production of some altered 30S ribosomes which are differentially sensitive to RNase.(2) The results presented below demonstrate that the molecules which cause suppression are tRNA. These observations justify the conclusions that the suppressor mutation did not occur in a structural gene for a ribosomal component, and that the decreased growth rate in rich medium, the increased sensitivity to streptomycin, and the production of altered 30S ribosomes are probably all secondary consequences of the suppressor mutation.