The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.     

Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching

Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents. Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism. Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain. Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site. Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching. The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites. Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching. This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme.     

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXIX. Impact of operating parameters in large-scale chromatography of proteins

Large-scale chromatography has been playing an important role in downstream treatment processing in biotechnology. In order to improve the productivity, the throughput of the chromatographic equipment was often increased by increasing the flow-rate and/or by increasing the column sample loading. This paper reports the results of a study on the impact of these and other operating parameters in affinity and ion-exchange chromatographic columns when used for protein purification. A sectional model was developed to predict protein adsorption processes in a packed column. The formulations of this mathematical model are presented in the Appendix. The present study was carried out with computer simulation based on this model and using data obtained from laboratory-scale columns. This model can simulate both the adsorption and washing stages of the protein purification process for both porous and non-porous particles. The effects of changing operating parameters were simulated and contour plots were generated for the easy identification of these effects. It was shown that both flow-rate and column loading can have a considerable impact on the processing rate and the yield of the column. As for the column capacity utilization, the impact of changing flow-rate is not significant at column loading of less than 80% in the test case. It was suggest that the present investigation provides a systematic predictive strategy which will greatly reduce the need for expensive, labour-intensive and time-consuming experimental work during process scale-up.     

Parasites of South African wildlife. XIV. Helminths of nyalas (Tragelaphus angasii) in the Mkuzi Game Reserve, KwaZulu-Natal

Reliability of event-related P3 parameters. Various parameters of the P3 wave were tested for reliability and test-retest stability. To this end, event-related P3 waves were evoked in 21 subjects during visual and auditory oddball tasks. The protocol was repeated one to two weeks later. Amplitude, latency and area between P3 and baseline were estimated by different procedures. The stability within and between sessions was assessed with the split-half method and a measure of test-retest reliability, respectively. A literature review revealed that latency estimation is most commonly done by computer-aided peak measurement, but determining peak parameters visually after interpellating superimposed artifacts turned out to be significantly more reliable. Interrater reliability for this visual inspection was very high if preestablished rules were employed. Amplitude and area were measured reliably by both computer-aided procedures and visual inspection. The area was quite stable both within and between sessions. Area can serve as a measure of cerebral activation and should therefore be determined. Based on the results recommendations are made about analyzing components of event-related potentials. The need to control for error variance is emphasized. This is the only way to improve methods enough that they will be suitable for use in single case diagnosis.     

On the usefulness of latent variable causal modeling in testing theories of naturally occurring events (including adolescent drug use): A rejoinder to Martin.

Replies to J. A. Martin's (see record 1983-06984-001) criticism of the present authors' (see record 1981-32589-001) methodology and conclusions. It is stated that Martin's criticisms are the result of misreading the present authors' paper and citations, whereas other criticisms were discussed in the original paper and resolved through alternate forms of data analysis. Further analyses are presented to address issues raised by Martin, and arguments against latent variable models are refuted. (14 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Peptides by activation of amino acids with CO on (Ni,Fe)S surfaces: implications for the origin of life

In experiments modeling volcanic or hydrothermal settings amino acids were converted into their peptides by use of coprecipitated (Ni,Fe)S and CO in conjunction with H2S (or CH3SH) as a catalyst and condensation agent at 100 degreesC and pH 7 to 10 under anaerobic, aqueous conditions. These results demonstrate that amino acids can be activated under geochemically relevant conditions. They support a thermophilic origin of life and an early appearance of peptides in the evolution of a primordial metabolism.     

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities in the sera and milk of cows with naturally occurring coliform mastitis

The presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities was determined in milk and serum of cows with naturally occurring coliform mastitis (CFM). TNF-alpha was detected in the sera from 26 of 32 cows with CFM. TNF-alpha levels were higher in the sera than in the milk. IL-6 was high in the sera of surviving CFM animals, but was low in animals that died and in healthy controls. Furthermore, the mean level of IL-6 was 20-fold higher in the milk than in the sera of mastitic cows. The level of IL-6 in the serum was correlated to that in the milk in individual animals. The presence of IL-6 and TNF-alpha in the sera appears to relate to severe clinical condition of CFM, in the milk whereas they may play a role in generating inflammation of the mammary gland.     

Sarcocystis buffalonis n.sp. (Protozoa: Sarcocystidae) from the water buffalo (Bubalus bubalis) in Vietnam

Sarcocystis buffalonis n. sp. is proposed for a species forming thick-walled, macroscopic sarcocysts in skeletal muscles and the esophagus of the water buffalo (Bubalus bubalis). Sarcocysts of S. buffalonis were found in 68 (10.5%) of 647 buffalo carcasses examined grossly at slaughter in Ho Chi Minh City in southern Vietnam. Sarcocystis buffalonis sarcocysts were 1-8 mm long and 0.1-0.5 mm wide. The cyst wall was 3-7.7 microns thick and had palisadelike villar protrusions that were constricted at the base, expanded laterally in the mid-region, and tapered distally. The villar protrusions contained microfilaments and electron-dense granules. Sarcocysts of Sarcocystis fusiformis, the other well-known macroscopic species occurring in water buffalo, were also found in 60 of the 68 animals infected with S. buffalonis. Sarcocysts of S. fusiformis were thin walled and had characteristic cauliflowerlike villar protrusions. Two of 7 cats fed isolated S. buffalonis sarcocysts were found to have 12 x 8 microns sporocysts in their intestine or feces 10 days after inoculation.     

Cytogenic studies of Hynobiidae (Urodela). XIV. Analysis of the chromosome of a Chinese salamander, Batrachuperus pinchonii (David)

The effects of beclomethasone dipropionate on pulmonary function and arterial blood gas values were investigated in horses with chronic obstructive pulmonary disease (COPD). Six mature mares, diagnosed as having COPD based on clinical signs, cytological examination of bronchoalveolar lavage and pulmonary function testing, were used. Beclomethasone dipropionate (3750 microg) was administered b.i.d. for a 2 week period with a metered dose inhaler using a mask. Pulmonary function tests and arterial blood gas analyses were performed at weekly intervals, starting before beclomethasone administration and for 4 weeks thereafter. Upper airway endoscopy and nasopharyngeal fungal cultures were performed before and after treatment. Maximal variations in transpulmonary pressure (deltaPL) were elevated in all horses at baseline. Beclomethasone administration resulted in a significant decrease in deltaPL in 5 horses, and deltaPL fell to within the normal range in 4 horses. Two weeks after the end of treatment, deltaPL was at or above baseline values in all horses. Total pulmonary resistance and elastance decreased significantly during treatment and returned to or above baseline values after the administration of beclomethasone was discontinued. At baseline, PaO2 range was 53-90 mmHg. In 4 horses with pronounced laboured breathing, PaO2 increased with treatment. One horse became reluctant to inhale the beclomethasone after one week, and only a transient improvement in respiratory function was noted in this animal. One horse developed a mild lower airway infection 24 h after the beginning of treatment, but no other possible side effects were noticed. Pharyngeal fungal cultures were negative before and after treatment. It can be concluded from the results of this study that inhaled beclomethasone dipropionate causes a marked improvement of respiratory function in horses with COPD.     

A carboxy-terminal truncation of 99 amino acids resulting from a novel mutation (Arg555-->stop) in the CHM gene leads to choroideremia

The gene content of the MHC class I telomerically adjacent region, in linkage disequilibrium with hereditary hemochromatosis, has not been well characterized yet. In the present work, we established three bacterial clone contigs, including mainly P1-derived artificial chromosomes. These contigs cover 89% of the 1.2-Mb 6p-subtelomeric region encompassing locus D6S105. Terminal exon trapping was applied to selected clones from these contigs. Forty-six independent terminal exons were identified and mapped within the region, 2 of which matched perfectly to expressed sequence tags. These 3' exons are all expressed in human fetal brain but differentially expressed in four tissues and two cell lines. The high number of exons identified indicates that the high gene density observed in the MHC class I region extends to this telomerically adjacent region.     

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.     

Synergistic effects in the melting of DNA hydration shell: melting of the minor groove hydration spine in poly(dA).poly(dT) and its effect on base pair stability

We propose that water of hydration in contact with the double helix can exist in several states. One state, found in the narrow groove of poly(dA).poly(dT), should be considered as frozen to the helix, i.e., an integral part of the double helix. We find that this enhanced helix greatly effects the stability of that helix against base separation melting. Most water surrounding the helix is, however, melted or disassociated with respect to being an integral part of helix and plays a much less significant role in stabilizing the helix dynamically, although these water molecules play an important role in stabilizing the helix conformation statically. We study the temperature dependence of the melting of the hydration spine and find that narrow groove nonbonded interactions are necessary to stabilize the spine above room temperature and to show the broad transition observed experimentally. This calculation requires that synergistic effects of nonbonded interactions between DNA and its hydration shell affect the state of water-base atom hydrogen bonds. The attraction of waters into narrow groove tends to retain waters in the groove and compress or strain these hydrogen bonds.