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PubMed2007

Iron acquisition in the dental pathogen Actinobacillus actinomycetemcomitans: what does it use as a source and how does it get this essential metal?

Actinobacillus actinomycetemcomitans requires iron to grow under limiting conditions imposed by synthetic and natural chelators. Although none of the strains tested used hemoglobin, lactoferrin or transferrin, all of them used FeCl3 and hemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, hemoglobin, and hemin but not transferrin. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was sensitive to proteinase K treatment. A. actinomycetemcomitans harbors the Fur-regulated afeABCD locus coding for iron acquisition in isogenic and non-isogenic cell backgrounds. The genome of this oral pathogen also harbors several other predicted iron uptake genes including the hitABC locus, which restored iron acquisition in the E. coli 1017 ent mutant. However, the disruption of this locus in the parental strain did not affect iron acquisition as drastically as the inactivation of AfeABCD, suggesting that the latter system could be more involved in iron transport than the HitABC system. The genome of this oral pathogen also harbors an active copy of the exbBexbDtonB operon, which could provide the energy needed for hemin acquisition. However, inactivation of each coding region of this operon did not affect the hemin and iron acquisition phenotypes of isogenic derivatives. This observation suggests that the function of these proteins could be replaced by those coded for by tolQ, tolR and tolA as it was described for other bacterial transport systems. Interruption of a hasR homolog, an actively transcribed gene that is predicted to code for an outer membrane hemophore receptor protein, did not affect the ability of an isogenic derivative to bind and use hemin under chelated conditions. This result also indicates that A. actinomycetemcomitans could produce more than one outer membrane hemin receptor as it was described in other human pathogens. All strains tested formed biofilms on plastic under iron-rich and iron-chelated conditions. However, smooth strains attached poorly and formed weaker biofilms when compared with rough isolates. The incubation of rough cells in the presence of FeCl3 or hemin resulted in an increased number of smaller aggregates and microcolonies as compared to the fewer but larger aggregates formed when cells were grown in the presence of dipyridyl.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

A domain mutations in 65 haemophilia A families and molecular modelling of dysfunctional factor VIII proteins.

A variety of mutations are found in haemophilia A families. Those with circulating, dysfunctional protein can provide insights into structural determinants of factor VIII function. A molecular model based upon the crystal structure of the homologous A domains in caeruloplasmin enables predictions of molecular consequences of mutations. To identify haemophilic mutations in coding regions for three A domains of factor VIII and predict amino acid substitutions important for coagulant cofactor function, amplified DNA fragments from 188 unrelated haemophilia A families were screened for heteroduplex formation. Exons 1-19 were examined. 65 families were positive for 58 distinct mutations (39 novel) on DNA sequencing. 12 were non-missense mutations. 38 missense mutations were found in patients that circulate or potentially circulate dysfunctional factor VIII protein and are in an A domain molecular model. Of these 38, 12 have identical residues among all known species of factors V, VIII and caeruloplasmin. These 38 mutations have been localized onto a factor VIII A domain molecular model. Of these, 19 are in coiled, 15 in beta-pleated sheet, and two each in turns and alpha-helical structures. 15 substituted residues are on the surface, nine are partially on the surface and 14 are buried within the model structure. Mutant side-chain substitutions were inserted to predict changes in surface groups or, for buried residues, potential surface areas whose structure is probably disrupted by the substitution.

Amino Acid Substitution↗
PubMedDate not supplied

[Statistical characteristics in primary structures of functional regions of Escherichia coli genome. II. Non-stationary Markov chains].

We introduced non-stationary Marcov chains for statistical description of the DNA E. coli structural domains. The values of all needed parameters for those chains was determined by the preliminary statistical processing of a wide set of the E. coli coding regions. It was shown that non-stationary models predict frequencies of occurrences of various combinations of nucleotides within the coding fragments of DNA, better than stationary ones. In particular non-stationary models give good approximation for short and long distance arrangement of nucleotides in the coding regions. The correlation parameters for neighbour codons and for neighbour amino acid residuals in E. coli protein's primary structure was determined from the non-stationary model of the second order. With the aid of the statistical criteria it was found that neighbour residuals in polypeptide chains can't be considered as independent. The new model of the DNA structural domain may be used in computer algorithms for recognition and classification of DNA functional regions.

Base Sequence↗
PubMed1985

Structure of the black beetle virus genome and its functional implications.

The black beetle virus (BBV) is an isometric insect virus whose genome consists of two messenger-active RNA molecules encapsidated in a single virion. The nucleotide sequence of BBV RNA1 (3105 bases) has been determined, and this, together with the sequence of BBV RNA2 (1399 bases) provides the complete primary structure of the BBV genome. The RNA1 sequence encompasses a 5' non-coding region of 38 nucleotides, a coding region for a protein of predicted molecular weight 101,873 (protein A, implicated in viral RNA synthesis) and a 3' proximal region encoding RNA3 (389 bases), a subgenomic messenger RNA made in infected cells but not encapsidated into virions. The RNA3 sequence starts 16 bases inside the coding region of protein A and contains two overlapping open reading frames for proteins of molecular weight 10,760 and 11,633, one of which is believed to be protein B, made in BBV-infected cells. A limited homology exists between the sequences of RNA1 and RNA2. Sequence regions have been identified that provide energetically favorable bonding between RNA2 and RNA1 possibly to facilitate their common encapsidation, and between RNA2 and negative strand RNA1 possibly to regulate the production of RNA3.

Base Sequence↗
PubMed1985

The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence.

We have determined the nucleotide sequence of a 4222-base segment of DNA which contains the promoter, the coding region, and the terminator of a crystal protein gene cloned from a Bacillus thuringiensis plasmid. A sequence of 1176 amino acids encoding a Mr 133,500 peptide was deduced from the single open reading frame. This protein-coding region was analyzed for codon usage, predicted hydropathy, and predicted secondary structure. Examination of the base sequence revealed the presence of several inverted and direct repeats located in both the coding and noncoding regions. S1 nuclease mapping was used to locate the transcription termination point at a site following a potentially very stable stem-and-loop structure.

Amino Acid Sequence↗
PubMed1997

Cloning and sequence analysis of a non-structural gene of an aquareovirus.

The nucleotide and deduced amino acid sequence of genome segment 11 encoding a nonstructural protein of an aquareovirus strain SBR have been determined. Nucleotide sequence analysis showed that the genome segment 11 of SBR virus is 780 nucleotides long and contains a major open reading frame that codes for a polypeptide of 236 amino acids with a predicted molecular weight of 25,504 Da. The second reading frame of genome segment 11 was 480 nucleotides long and codes for a polypeptide of 145 with a predicted molecular weight of 15,715 Da. The genome segment 11 contains 24 nontranslated nucleotides at the 5'-end and 48 nontranslated nucleotides at the 3'-end. This gene codes for two nonstructural polypeptides NS29 and NS15. Comparison of the deduced amino acid sequence of this gene with the published sequences of other members of the family Reoviridae indicated no sequence relatedness.

Amino Acid Sequence↗
PubMed2006

Identification of a non-mammalian leptin-like gene: characterization and expression in the tiger salamander (Ambystoma tigrinum).

Leptin is well established as a multifunctional cytokine in mammals. However, little is known about the evolution of the leptin gene in other vertebrates. A recently published set of ESTs from the tiger salamander (Ambystoma tigrinum) contains a sequence sharing 56% nucleotide sequence identity with the human leptin cDNA. To confirm that the EST is naturally expressed in the salamander, a 409bp cDNA was amplified by RT-PCR of salamander testis and stomach mRNAs. The coding sequence of the cDNA is predicted to encode 169 amino acids, and the mature peptide to consist of 146 residues, as in mammals. Although the overall amino acid identity with mammalian leptins is only 29%, the salamander and mammalian peptides share common structural features. An intron was identified between coding exons providing evidence that the sequence is present in the salamander genome. Phylogenetic analysis showed a rate of molecular divergence consistent with the accepted view of vertebrate evolution. The pattern of tissue expression of the leptin-like cDNA differed between metamorphosed adult individuals of different sizes suggesting possible developmental regulation. Expression was most prominent in the skin and testis, but was also detected in tissues in which leptin mRNA is present in mammals, including the fat body, stomach, and muscle. The characterization of a salamander leptin-like gene provides a basis for understanding how the structure and functions of leptin have altered during the evolution of tetrapod vertebrates.

Ambystoma↗
PubMed2004

Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis.

The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population of linear DNA molecules that are heterogeneous in size. The length of the shortest molecules is 30,922 bp, whereas the longer molecules have expanded terminal tandem arrays (nx738 bp). The mitochondrial genome is highly compact, with less than 8% of the sequence corresponding to non-coding intergenic spacers. In silico analysis predicted genes encoding fourteen protein subunits of complexes of the respiratory chain and ATP synthase, rRNAs of the large and small subunits of the mitochondrial ribosome, and twenty-four transfer RNAs. These genes are organized into two transcription units. In addition, six intronic ORFs coding for homologues of RNA maturase, reverse transcriptase and DNA endonucleases were identified. In contrast to its overall molecular architecture, the coding sequences of the linear mitochondrial DNA of C. parapsilosis are highly similar to their counterparts in the circular mitochondrial genome of its close relative C. albicans. The complete sequence has implications for both mitochondrial DNA replication and the evolution of linear DNA genomes.

Base Sequence↗
PubMed1994

Detection of mixed modulation using correlated and uncorrelated noise modulators.

This article is concerned with the mechanisms underlying the detection of amplitude modulation (AM), frequency modulation (FM), and mixed modulation (MM), i.e., simultaneously occurring AM and FM. In a previous study [B. C. J. Moore and A. Sek, J. Acoust. Soc. Am. 92, 3119-3131 (1992)], psychometric functions were measured for the detection of AM alone and FM alone, using a 10-Hz sinusoidal modulator and a 1-kHz carrier frequency. Detectability was then measured for combined AM and FM, with modulation depths selected so that each type of modulation would be equally detectable if presented alone. The detectability of the MM was better than would be predicted if the two types of modulation were coded completely independently. This study examined the possibility that the good detectability of MM was caused by the fact that the AM and the FM were correlated, so that each was predictable from the other. The design was similar to that of our earlier study, but the 10-Hz sinusoidal modulator was replaced by a narrow-band noise modulator. In the MM conditions, the modulators for AM and FM were either strongly positively correlated or essentially uncorrelated. In experiment 1, the waveforms of the noise modulators were fixed throughout the experiment (frozen noise). In experiment 2, the waveforms of the noise modulators were chosen independently for each trial. In both experiments, for both correlated and uncorrelated modulators, the detectability of the MM was better than would be predicted if the two types of modulation were coded completely independently.(ABSTRACT TRUNCATED AT 250 WORDS)

Acoustic Stimulation↗
PubMed2002

Spatial structure and symmetry of simple-cell receptive fields in macaque primary visual cortex.

I present measurements of the spatial structure of simple-cell receptive fields in macaque primary visual cortex (area V1). Similar to previous findings in cat area 17, the spatial profile of simple-cell receptive fields in the macaque is well described by two-dimensional Gabor functions. A population analysis reveals that the distribution of spatial profiles in primary visual cortex lies approximately on a one-parameter family of filter shapes. Surprisingly, the receptive fields cluster into even- and odd-symmetry classes with a tendency for neurons that are well tuned in orientation and spatial frequency to have odd-symmetric receptive fields. The filter shapes predicted by two recent theories of simple-cell receptive field function, independent component analysis and sparse coding, are compared with the data. Both theories predict receptive fields with a larger number of subfields than observed in the experimental data. In addition, these theories do not generate receptive fields that are broadly tuned in orientation and low-pass in spatial frequency, which are commonly seen in monkey V1. The implications of these results for our understanding of image coding and representation in primary visual cortex are discussed.

Animals↗
PubMed2002

A comparison of transient dose model predictions and experimental measurements.

The RADTRAN and RISKIND transportation risk analysis computer codes are the primary tools used to estimate dose consequences and risks associated with the transport of radioactive material. Over the years, some of the mathematical models used within the two computer codes have been updated and the methodologies to calculate input parameters have been improved. In addition, both codes have been evaluated for ease of use and appropriateness of application and verified against other computer codes that perform similar calculations. However, neither code has been validated against experimental data. This report discusses the results of five sets of experimental measurements used to partially validate the specific mathematical models used to predict the dose to an individual due to a passing shipment of radioactive material within the RADTRAN and RISKIND computer codes. Based on the comparisons it was found that RISKIND most closely predicted the measured dose in the majority of the investigated scenarios and that 12 out of 14 cases demonstrate the expected inverse relationship between the measured dose and the distance of closest approach. Only half of the data demonstrated the expected inverse relationship between dose and speed of travel.

Algorithms↗
PubMed2001

Information transfer between rhythmically coupled networks: reading the hippocampal phase code.

There are numerous reports on rhythmic coupling between separate brain networks. It has been proposed that this rhythmic coupling indicates exchange of information. So far, few computational models have been proposed that explore this principle and its potential computational benefits. Recent results on hippocampal place cells of the rat provide new insight; it has been shown that information about space is encoded by the firing of place cells with respect to the phase of the ongoing theta rhythm. This principle is termed phase coding and suggests that upcoming locations (predicted by the hippocampus) are encoded by cells firing late in the theta cycle, whereas current location is encoded by early firing in the theta cycle. A network reading the hippocampal output must inevitably also receive an oscillatory theta input in order to decipher the phase-coded firing patterns. In this article, I propose a simple physiologically plausible mechanism implemented as an oscillatory network that can decode the hippocampal output. By changing only the phase of the theta input to the decoder, qualitatively different information is transferred: the theta phase determines whether representations of current or upcoming locations are read by the decoder. The proposed mechanism provides a computational principle for information transfer between oscillatory networks and might generalize to brain networks beyond the hippocampal region.

Action Potentials↗
PubMed1987

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.

Amino Acid Sequence↗
PubMed2002

Physicochemical methods for predicting the biological potency of recombinant follicle stimulating hormone: an international collaborative study of isoelectric focusing and capillary zone electrophoresis.

Two methods for predicting the specific in vivo bioactivity of recombinant follicle stimulating hormone (FSH), based on quantitative measures of isoform distribution by isoelectric focusing (IEF)(1) and by capillary zone electrophoresis (CZE)(2) respectively, have been subjected to an international collaborative study by six laboratories from six countries. Both methods were used to estimate the predicted bioactivities of four preparations of follitropin beta, coded FSH A-D, differing widely in their isoform compositions and specific bioactivities. The mean predicted estimate of potency by IEF and CZE for each FSH preparation by each laboratory was within 80-125% of its potency estimated by bioassay, except for the mean estimates by CZE of that for FSH A by one laboratory and of that of FSH D by another. Each of the six laboratories using the IEF method, and each of the five laboratories using the CZE method were able to rank these FSHs according to their bioactivities, namely FSH B>FSH C>FSH A>FSH D. All laboratories were able to use both IEF and CZE to discriminate between FSH A and C, with bioactivities within 76-132% of one another. Four of six laboratories were able to use IEF, and two of five laboratories were able to use CZE, to discriminate between FSH B and C, with bioactivities within 89-112% of one another. This suggests that the accuracy and precision of both these methods should be sufficient to discriminate between FSHs which would meet or fail European Pharmacopoeia requirements for this type of hormone, since these stipulate that estimates of potency should fall between 80-125% of its stated potency. Using in most cases duplicate estimates in two independent assays, and excluding Laboratory 4, the pooled intra-laboratory geometric coefficient of variation (GCV) was about 4% for both IEF and CZE, and the inter-laboratory GCV was about 7% for IEF and about 10% for CZE. The use of one FSH preparation as a standard, with its specific activity as an assigned value, reduced the inter-laboratory variability of estimates for the remaining FSHs by both methods. This increased the accuracy of the predicted estimates of bioactivity for these remaining FSHs in terms of their approximation to the values for their bioactivities estimated by bioassay. These data therefore suggest that both these methods, and particularly IEF, are sufficiently accurate, precise and robust to be used for predicting the bioactivity of batches of follitropin beta, and especially if used with a standard preparation.

Chemical Phenomena↗
PubMed2001

The low-incidence MNS antigens M(v), s(D), and Mit arise from single amino acid substitutions on GPB.

BACKGROUND: GPB carries 'N' at its N:-terminus and S and s, determined by a polymorphism at amino acid position 29 (Met29Thr). The low-incidence antigens M(v), s(D), and Mit are associated with weakened expression of S and/or s, and the purpose of this study was to define their molecular bases. METHODS: The GPB gene (GYPB) was sequenced after RT-PCR of RNA from four samples: two M(v)+, one s(D)+, and one Mit+. The point mutations observed were confirmed by sequencing of genomic DNA from these and other examples of s(D)+ and Mit+ samples. RESULTS: A point mutation of 65C>G observed in the M(v)+ samples predicted a change of Thr3Ser. A mutation of C>G at nucleotide 173 of the GYPB coding sequence, observed in two s(D)+ samples, predicted a change of Pro39Arg. Three Mit+ samples showed a nucleotide substitution of 161G>A, which predicted a change of Arg35His. Altered expression of S or s was confirmed by serologic tests. CONCLUSION: These results confirm that Arg35 is important for full expression of S. Pro39 and, surprisingly, Thr3 are also important for full expression of s. Furthermore, Thr3 must be essential for expression of 'N,' as M(v)+ RBCs lack 'N.'

Amino Acid Substitution↗
PubMed2003

Phylogenetic network and physicochemical properties of nonsynonymous mutations in the protein-coding genes of human mitochondrial DNA.

Theories on molecular evolution predict that phylogenetically recent nonsynonymous mutations should contain more non-neutral amino acid replacements than ancient mutations. We analyzed 840 complete coding-region human mitochondrial DNA (mtDNA) sequences for nonsynonymous mutations and evaluated the mutations in terms of the physicochemical properties of the amino acids involved. We identified 465 distinct missense and 6 nonsense mutations. 48% of the amino acid replacements changed polarity, 26% size, 8% charge, 32% aliphaticity, 13% aromaticity, and 44% hydropathy. The reduced-median networks of the amino acid changes revealed relatively few differences between the major continent-specific haplogroups, but a high variation and highly starlike phylogenies within the haplogroups. Some 56% of the mutations were private, and 25% were homoplasic. Nonconservative changes were more common than expected among the private mutations but less common among the homoplasic mutations. The asymptotic maximum of the number of nonsynonymous mutations in European mtDNA was estimated to be 1,081. The results suggested that amino acid replacements in the periphery of phylogenetic networks are more deleterious than those in the central parts, indicating that purifying selection prevents the fixation of some alleles.

Africa↗
PubMed1995

Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR.

Recently, we identified and cloned a human endothelial cell protein C/activated protein C receptor (EPCR). EPCR was predicted to be a type 1 transmembrane glycoprotein and a novel member of the CD1/major histocompatibility complex superfamily with 28% identity with CD1d. Even greater homology (62% identity) was detected with the murine protein, CCD41, which was previously characterized as a centrosome-associated, cell cycle-dependent protein. This raised the possibility that CCD41 was the murine homologue of EPCR. To address this possibility, to better understand structure-function relationships, and to facilitate physiological experiments on EPCR function, we cloned and sequenced murine and bovine EPCR from endothelial cell cDNA libraries. The nucleotide sequence of murine EPCR and CCD41 exhibited five differences corresponding to one base change, three single-base insertions, and one base deletion in the protein coding region. As a result, the predicted structures of EPCR and CCD41 differed in their amino and carboxyl termini but were identical in the central portion of the coding sequence. Based on comparison of the murine, bovine, and human EPCR sequences and the regions where discrepancies between murine EPCR and CCD41 were detected, we believe that CCD41 is probably identical to murine EPCR and that the reported sequence differences are likely the result of compression on the sequencing gel. Compared with human EPCR, the murine and bovine sequences were 69 and 73% identical, respectively, and 57% of the residues were identical between all three species. Both bovine and murine EPCR could bind human activated protein C when the cDNA clones were transfected into 293T cells. Like human EPCR, of the cell lines tested, the murine EPCR message was restricted to endothelium. Cloning of the murine and bovine homologue of EPCR will facilitate in vivo and in vitro studies of the role of EPCR in the protein C pathway.

Amino Acid Sequence↗
PubMed1984

Utility of the MMPI-168 with adolescents.

Investigated the utility of the MMPI-168 with an adolescent psychiatric sample of 110 Ss by comparing published conversion values for adults to population-specific derived equations developed through both regression and substitution methods. Data analyses for the three equations compared 168 T scores to long-form scores via correlation coefficients and t-tests of mean scale score differences. The ability of the 168 to make accurate decisions with regard to profile validity, degree of elevation, high points, code typing, and diagnosis also was examined. Results that used the published equations were similar to those of previous 168 studies with adults. Although population-specific conversion equations produced means and standard deviations that more closely approximated the full MMPI, they failed to produce higher T score correlations or better prediction of profile validity and code-types than the published values. Appropriate uses and limitations of the 168 with adolescents are discussed.

Adolescent↗