Diffuse axonal injury induced by simultaneous moderate linear and angular head accelerations in rats.

Diffuse axonal injury (DAI) is one of the most common and important pathologic features of human traumatic brain injury (TBI), accounting for high mortality and development of persistent post-traumatic neurologic sequelae. Although a relatively high number of therapies have been shown to be effective in experimental models, there are currently few treatments that are effective for improving the prognosis of clinical DAI. A major reason is the failure of current models to validly reproduce the pathophysiological characteristics observed after clinical DAI. In the present study, we employed a specially designed, highly controllable model to induce a sudden rotation in the coronal plane (75 degrees rotation at 1.6x10(4) degrees/s) combined with lateral translation (1.57 cm displacement at 3.4x10(2) cm/s) to the rat's head. We were interested in discovering whether the combined accelerations could reproduce the pathophysiological changes analogous to those seen in human DAI. The axonal injury as assessed with amyloid protein precursor (APP) as a marker was consistently present in all injured rats. The commonly injured brain regions included the subcortical regions, deep white matter, corpus callosum and brain stem. The evolution of APP accumulations in brain sections depicted the detailed progression of axonal pathology. Ultrastructural studies gave further insights into the presence and progression of axonal injury. All injured rats exhibited transient physiological dysfunction, as well as immediate and dramatic neurological impairment that still persisted at 14 days after injury. These results suggest that this model reproduced the major pathophysiological changes analogous to those observed after severe clinical TBI and provides an attractive vehicle for experimental brain injury research.

Determining divergence times with a protein clock: update and reevaluation.

A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.

Converting amino acid alignment scores into measures of evolutionary time: a simulation study of various relationships.

Amino acid substitution tables are essential for the proper alignment of protein sequences, and alignment scores based on them can be transformed into distance measures by various means. In the simplest case, the negative log of the score is used. This Poisson relationship assumes that all sites are equally likely to change, however. A more accurate relationship would correct for different rates of change at each residue position. Recently, Grishin (J. Mol. Evol. 41:675-679, 1995) published a set of simple equations that correct for various circumstances, including different rates of change at different sites. We have used these equations in conjunction with similarity scores that take into account constraints on amino acid interchange. Simulation studies show a linear relationship between these calculated distances and the numbers of allowed mutations based on the observed variation of rate at all sites in various proteins.

Determining divergence times of the major kingdoms of living organisms with a protein clock.

Amino acid sequence data from 57 different enzymes were used to determine the divergence times of the major biological groupings. Deuterostomes and protostomes split about 670 million years ago and plants, animals, and fungi last shared a common ancestor about a billion years ago. With regard to these protein sequences, plants are slightly more similar to animals than are the fungi. In contrast, phylogenetic analysis of the same sequences indicates that fungi and animals shared a common ancestor more recently than either did with plants, the greater difference resulting from the fungal lineage changing faster than the animal and plant lines over the last 965 million years. The major protist lineages have been changing at a somewhat faster rate than other eukaryotes and split off about 1230 million years ago. If the rate of change has been approximately constant, then prokaryotes and eukaryotes last shared a common ancestor about 2 billion years ago, archaebacterial sequences being measurably more similar to eukaryotic ones than are eubacterial ones.

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin. A new member of the serpin superfamily of proteins from insects.

The cDNA of silkworm (Bombyx mori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature sw-Achy begins with Phe1 and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human alpha-1-antitrypsin and human alpha-1-antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with alpha-chymotrypsin was identified as Thr343-Ser344 by direct amino-acid sequence analysis of cleaved and purified protein.

Evolution by acquisition: the case for horizontal gene transfers.

One of the most debated questions in the field of molecular evolution is the possible role of horizontal transfer in evolution. Of all the claims that have been made over the years, those reporting transfers between eukaryotes and prokaryotes are the most controversial. Here we present the cases for and against several such possible gene acquisitions.

Tracing the origin of retroviruses.

Reverse transcriptase sequences, which are fundamental to retrovirus existence, are widely distributed in the living world. Phylogenies based on their sequences set vertebrate retroviruses apart as relatively modern creations. Their nearest evolutionary relatives are a large group of transposable elements that have all the standard retrovirus equipment except spliced envelope proteins. The distribution of these elements suggests a long-standing presence predating the radiation of plants, fungi, and animals. There is another large group of elements, LINEs, that also contain recognizable reverse transcriptase sequences and which likely diverged even earlier, as evidenced by their presence in trypanosomes and other protists. They lack tRNA priming sites--which they could have lost--but they do exhibit characteristic eukaryotic polyadenylation. These elements are problematic in that the sequences are so degenerate in most instances that it is not possible to identify the accessory enzymes or structural proteins with any confidence, leaving major gaps in our reconstruction of events. Even with these gaps, however, the historical beginnings of retroviruses can be traced back to events coincident with the prokaryotic invasion of primitive eukaryotes.

A naturally occurring horizontal gene transfer from a eukaryote to a prokaryote.

Naturally occurring horizontal gene transfers between nonviral organisms are difficult to prove. Only with the availability of sequence data from a wide variety of organisms can a convincing case be made. In the case of putative gene transfers between prokaryotes and eukaryotes, the minimum requirements for inferring such an event include (1) sequences of the transferred gene or its product from several appropriately divergent eukaryotes and several prokaryotes, and (2) a similar set of sequences from the same (or closely related organisms) for another gene or genes. Given these criteria, we believe that a strong case can be made for Escherichia coli having acquired a second glyceraldehyde-3-phosphate dehydrogenase gene from some eukaryotic host. Ancillary observations on the general rate of change and the time of the prokaryote-eukaryote divergence support the notion.

Construction of a facsimile data set for large genome sequence analysis.

A test was devised for exploring the question of whether it will be possible to identify genes in large-scale genome studies solely by sequence comparison with current sequence collections. To this end, a facsimile data set was constructed by dividing GenBank Release 56 randomly into two halves, one to serve as a reference set and the other intended to simulate raw data anticipated from large genome sequence projects. All supplementary information and identifying marks were removed from the test set after assignment of random identification numbers to each entry and their encryption. Because noncoding intervening sequences (introns) are underrepresented in GenBank, a program that introduced (simulated) introns into mRNA and prokaryotic sequences was devised. In a further attempt to make the problem of identification more realistic, random base substitutions and single-base deletions were also incorporated. The randomly ordered entries were concatenated, along with random intergenic flanking sequences, into a single long "chromosome" 33 Mb in length and then cut into "cosmids" 50-100 kb long. The chopping process was conducted in such a way that terminal overlaps would allow the order of the entries in the chromosome to be reconstituted. Finally, the sequences of a substantial fraction of the cosmids were converted to their complements. Preliminary searching of 10 test cosmids revealed that more than two-thirds of the entries in the test set should be readily identifiable by type of gene product solely on the basis of comparison with the reference set. These preliminary results suggest that existing computer regimens and sequence collections would be able to identify the majority of eukaryotic genes in any new raw data set, the existence of introns not withstanding. Moreover, the analysis can be conducted in pace with the data collection so that the search results and summary identifications will be instantly available to the research community at large.

Retrovirus phylogeny and evolution.

The elucidation of complete genomic sequences from a wide variety of retroviruses and retrotransposons has allowed the construction of sequence-based phylogenies that reveal their evolutionary history. True retroviruses, whether exogenous or endogenous, tend to cluster into four major groups. Not only is there no distinction between exogenous and endogenous viruses, but their evolutionary limb lengths on the phylogenetic trees are comparable. This can be taken as evidence favoring a dynamic equilibrium balancing a constant invasion of germlines by infectious retroviruses on the one hand, with subsequent escape of endogenous viruses to alternative hosts on the other. Retroviruses share a common ancestry with a wide variety of retrotransposons and other reverse transcriptase-bearing entities. One of these retrotransposon groups, the Gypsy group, resembles the Moloney mouse group of retroviruses much more closely than it does other retroviruses. The simplest explanation is that the evolutionary rate of the retrotransposon is much slower than the retrovirus rate and that among the retroviruses the Moloney mouse group has been evolving more slowly than the other three groups, leaving the two short-limbed taxa more similar. The alternative explanation that these two groups actually shared a common ancestor more recently than has either with the other retrovirus groups is not supported by residue-by-residue character assessment.

Origins and evolutionary relationships of retroviruses.

As is the case for some other RNA viruses, the amino acid sequences of retroviral proteins change at an astonishing rate. For example, the proteases of the human immunodeficiency virus (HIV) and the visna lentivirus with which it is often compared are as different as the proteases of fungi and mammals, and those of the human type I leukemia virus are as different from HIV or visna as are the proteins of humans and bacteria. That the sequences of retrovirus proteins can be recognized as sharing common ancestry with non-retroviral proteins implies that the vastly accelerated change has begun only recently or occurs very sporadically. Only a scheme whereby exogenous retroviruses exist as short-lived bursts upon a backdrop of germline-encoded endogenous viruses is consistent with the sequence data. Retroviruses are related to many other reverse transcriptase-bearing entities present in the genomes of eukaryotes. They also have proteins that are homologous with those of some plant and animal DNA viruses, and their reverse transcriptase is recognizably similar to sequences found in the introns of some fungal mitochondria. Computer alignment of all these sequences allows an overall phylogeny to be constructed that chronicles the history of events leading to infectious retroviruses.

Sequence comparisons of retroviral proteins: relative rates of change and general phylogeny.

The inferred amino acid sequences of 10 specific gene products from nine retroviruses were aligned by computer, all evolutionary distances between them calculated, and evolutionary trees constructed. Not unexpectedly, the various gene products are changing at different rates, the reverse transcriptase being the least and the envelope proteins the most different from one retrovirus to another. For the most part, trees based on the retroviral enzyme sequences are congruent, indicating that extensive genetic recombination has not been a major factor in the evolution of the central part of the genome. In the case of envelope protein sequences, however, the sequences clearly exhibit evidence of multiple cross-over events between quite distantly related retroviruses. A composite phylogenetic tree was constructed from the four retroviral enzyme sequences, and a number of important historical happenings were interpreted in the light of the time scale it affords.

Progressive sequence alignment as a prerequisite to correct phylogenetic trees.

A progressive alignment method is described that utilizes the Needleman and Wunsch pairwise alignment algorithm iteratively to achieve the multiple alignment of a set of protein sequences and to construct an evolutionary tree depicting their relationship. The sequences are assumed a priori to share a common ancestor, and the trees are constructed from difference matrices derived directly from the multiple alignment. The thrust of the method involves putting more trust in the comparison of recently diverged sequences than in those evolved in the distant past. In particular, this rule is followed: "once a gap, always a gap." The method has been applied to three sets of protein sequences: 7 superoxide dismutases, 11 globins, and 9 tyrosine kinase-like sequences. Multiple alignments and phylogenetic trees for these sets of sequences were determined and compared with trees derived by conventional pairwise treatments. In several instances, the progressive method led to trees that appeared to be more in line with biological expectations than were trees obtained by more commonly used methods.

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

A computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment that is homologous with the ribonuclease H of Escherichia coli. The segment occurs at the carboxyl terminus of the region assigned to the 90-kDa reverse transcriptase polypeptide. In contrast, a section nearer the amino terminus of this sequence can be aligned with nonretroviral polymerases. The order of activities in the pol gene is thus: polymerase-ribonuclease-endonuclease. On another note, all retroviral endonuclease sequences contain a consensus zinc-binding "finger." This should not be confused with the well-known zinc requirement of reverse transcriptases.