Systematic variation of amino acid substitutions for stringent assessment of pairwise covariation

During protein evolution, amino acids change due to a combination of functional constraints and genetic drift. Proteins frequently contain pairs of amino acids that appear to change together (covariation). Analysis of covariation from naturally occurring sets of orthologs cannot distinguish between residue pairs retained by functional requirements of the protein and those pairs existing due to changes along a common evolutionary path. Here, we have separated the two types of covariation by independently recombining every naturally occurring amino acid variant within a set of 15 subtilisin orthologs. Our analysis shows that in this family of subtilisin orthologs, almost all possible pairwise combinations of amino acids can coexist. This suggests that amino acid covariation found in the subtilisin orthologs is almost entirely due to common ancestral origin of the changes rather than functional constraints. We conclude that naturally occurring sequence diversity can be used to identify positions that can vary independently without destroying protein function.     

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.     

Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions

Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple pathways within a single organism leading to Cr(VI) reduction has not been reported previously.     

Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.     

Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth

Cr(VI) reduction was observed during tests with Shewanella oneidensis MR-1 (previously named S. putrefaciens MR-1) while being grown with nitrate or fumarate as electron acceptor and lactate as electron donor. From the onset of anoxic growth on fumarate, we measured a gradual and progressive increase in the specific Cr(VI) reduction rate with incubation time until a maximum was reached at late exponential/early stationary phase. Under denitrifying conditions, the specific Cr(VI) reduction rate was inhibited by nitrite, which is produced during nitrate reduction. However, once nitrite was consumed, the specific reduction rate increased until a maximum was reached, again during the late exponential/early stationary phase. Thus, under both fumarate- and nitrate-reducing conditions, an increase in the specific Cr(VI) reduction rate was observed as the microorganisms transition from oxic to anoxic growth conditions, presumably as a result of induction of enzyme systems capable of reducing Cr(VI). Although Cr(VI) reduction has been studied in MR-1 and in other facultative bacteria under both oxic and anoxic conditions, a transition in specific reduction rates based on physiological conditions during growth is a novel finding. Such physiological responses provide information required for optimizing the operation of in situ systems for remediating groundwater contaminated with heavy metals and radionuclides, especially those that are characterized by temporal variations in oxygen content. Moreover, such information may point the way to a better understanding of the cellular processes used by soil bacteria to accomplish Cr(VI) reduction.     

Constant variation in structure and function of geometrical isomers of acitretin under natural light

Acitretin, a beneficial retinoid, was shown to undergo constant structural interconversions among its geometrical isomers (all-trans-acitretin, 9-cis-acitretin, 13-cis-acitretin, 9, 13-di-cis-acitretin, etc.) by photoisomerization under natural light. The photoisomerization was zero order reaction with an apparent velocity of 4x107 M/min under illumination by white fluorescent lamps (1, 200 lx). An equilibrium mixture of the geometrical isomers (all-trans-acitretin 20%, 9-cis-acitretin 15%, 13-cis-acitretin 30%, 9, 13-di-cis-acitretin 15%, and unidentified compounds 20%) was formed at around 30 min. Equilibrium mixtures with similar composition were obtained by photoisomerization reactions starting from other geometrical isomers. Geometrical isomers of acitretin thus formed, showed different effects to induce differentiation of human acute promyelocytic leukemia cells (HL-60 cells): activity of all-trans-acitretin (ED50, 3.2 x 10(-6) M), 9-cis-acitretin (ED50, 2.3 x 10(-5)M), 13-cis-acitretin (ED50, 1.1 x 10(-5)M), 9, 13-di-cis-acitretin (ED50, 2.6 x 10(-6)M). 9-cis-Acitretin acted synergistically with all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin on HL-60 cells. On the other side, all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin acted additively. Geometrical isomers of acitretin showed different effects on differentiation of human epidermal keratinocytes; expression of keratinocyte differentiation markers, keratin 1 and keratin 10, were suppressed more strongly by 9-cis-acitretin and 13-cis-acitretin as compared to all-trans-acitretin or 9, 13-di-cis-acitretin.     

Effects of simultaneous exercise and loud music on hearing acuity and auditory function

Hearing acuity can be reduced temporarily after exposure to loud noise, and the physiological responses that occur with exercise may enhance this effect. Currently, it is not known whether short-term reductions in hearing acuity after noise exposure and exercise are a result of temporary changes in auditory function. Therefore, the purpose of this investigation was to determine the acute effects of simultaneous exercise and loud music on hearing acuity and auditory function in young, healthy women. Nine women (age = 22 +/- 5 years, body mass index = 23.9 +/- 2.2, Vo(2)peak = 30.6 +/- 6.0 ml x kg(-1) x min(-1)) with normal hearing thresholds (<20 dB hearing level) underwent each of 3 conditions in a randomized counterbalanced design: (a) loud music exposure of 90 to 95 dB sound pressure level for 20 minutes, (b) exercise at 60% Vo(2)peak on a cycle ergometer for 20 minutes, and (c) simultaneous exercise and music exposure for 20 minutes. Hearing acuity and auditory function were assessed via pure-tone hearing thresholds and distortion product otoacoustic emission amplitudes, respectively, at frequencies of 2, 3, 4, 6, and 8 kHz presented in random order before and after each condition. Results indicate that hearing acuity and auditory function remained unaltered after exposure to each condition (p > 0.05). These findings provide evidence that hearing acuity and auditory function in young women do not change after short-term exposure to moderate-intensity exercise and loud music. Thus, listening to loud music with earphones during moderate-intensity exercise does not pose acute hearing health concerns for young physically fit adults with normal hearing.     

Mathematical models of cochlear nucleus onset neurons: I. Point neuron with many weak synaptic inputs

The cochlear nucleus (CN) presents a unique opportunity for quantitatively studying input-output transformations by neurons because it gives rise to a variety of different response types from a relatively homogeneous input source, the auditory nerve (AN). Particularly interesting among CN neurons are Onset (On) neurons, which have a prominent response to the onset of sustained sounds followed by little or no response in the steady-state. On neurons contrast sharply with their AN inputs, which respond vigorously throughout stimuli. On neurons can entrain to stimuli (firing once per cycle of a periodic stimulus) at up to 1000 Hz, unlike their AN inputs. To understand the mechanisms underlying these response patterns, we tested whether an integrate-to-threshold point-neuron model with a fixed refractory period can account for On discharge patterns for tones, systematically examining the effect of membrane time constant and the number and strength of the exclusively excitatory AN synaptic inputs. To produce both onset responses to high-frequency tone bursts and entrainment to a broad range of low-frequency tones, the model must have a short time constant (0.125 ms) and a large number (>100) of weak synaptic inputs, properties that are consistent with the electrical properties and anatomy of On-responding cells. With these parameters, the model acts like a coincidence detector with a threshold-like relationship between the instantaneous discharge rates of the output and the inputs. Onset responses to high-frequency tone bursts result because the threshold effect enhances the initial response of the AN inputs and suppresses their relatively lower sustained response. However, when the model entrains across a broad range of frequencies, it also produces short interspike intervals at the onset of high-frequency tone bursts, a response pattern not found in all types of On neurons. These results show a tradeoff, that may be a general property of many neurons, between following rapid stimulus fluctuations and responding without short interspike intervals at the onset of sustained stimuli.     

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.