Identification of Major Phylogenetic Branches of Inhibitory Ligand-Gated Channel Receptors

The gene superfamily of ligand-gated ion channel (LGIC) receptors is composed of members of excitatory LGIC receptors (ELGIC) and inhibitory LGIC receptors (ILGIC), all using amino acids as ligands. The ILGICs, including GABA_A, Gly, and GluCl receptors, conduct Cl⁻ when the ligand is bound. To evaluate the phylogenetic relationships among ILGIC members, 90 protein sequences were analyzed by both maximum-parsimony and distance matrix-based methods. The strength of the resulting phylogenetic trees was evaluated by means of bootstrap. Four major phylogenetic branches are recognized. Branch I, called BZ, for the majority of the members are known to be related to benzodiazepine binding, is subdivided into IA, composed of all GABA_A receptor α subunits, and IB, composed of the γ and ε subunits, which are shown to be tightly linked. Branch II, named NB for non–benzodiazepine binding, and consisting of GABA_A receptor β, δ, π, and ρ subunits, is further subdivided into IIA, containing β subunits; IIB, containing δ, and π subunits; and IIC, containing ρ subunits. Branch IIIA, composed of vertebrate Gly receptors, is loosely clustered with Branch IIIB, composed of invertebrate GluCl receptors, to form Branch III, which is designated NA for being non–GABA responsive. Branch IV is called UD for being undefined in specificity. The existence of primitive forms of GABA_A receptor non-β subunits in invertebrates is first suggested by the present analysis, and the identities of sequences p25123 from Drosophila melanogaster, s34469 from Lymnaea stagnalis, and u14635 and p41849 from C. aenorhabditis elegans are determined to be different from their previously given annotations. The proposed branching classification of ILGICs provides a phylogenetic map, based on protein sequences, for tracing the evolutionary pathways of ILGIC receptor subunits and determining the identities of newly discovered subunits on the basis of their protein sequences. Received: 15 April 1997 / Accepted: 11 March 1998     

Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth

Surfactant protein A (SP-A) functions in homeostasis of lung surfactant and in innate immunity. SP-A is secreted by the fetal lung into amniotic fluid. Additionally it has been detected in gestational tissues. We propose that SP-A influences intrauterine inflammation that is commonly associated with preterm birth, the main underlying cause of neonatal mortality and morbidity. We used our previously established mouse model of LPS-induced preterm birth of live-born pups to investigate the role of SP-A in preterm birth. Mice overexpressing rat SP-A (rSP-A) under the control of human SP-C promoter were used. Cytokine concentrations in maternal and fetal serum and in amniotic fluid and mRNA levels of several inflammatory mediators in lungs and in intrauterine tissues were quantified using Cytometric Bead Array and RNase Protection Assay, respectively. Higher levels of SP-A mRNA were observed in fetal lungs and intrauterine tissues of rSP-A mice compared with wild-type. Using Western blot we detected excess of SP-A protein in fetal lung and in amniotic fluid of rSP-A animals. Despite some differences in the basal levels of TNF-α and IL-10 between rSP-A and wild-type animals, there were no differences in the duration of pregnancy. However, the levels of TNF-α, IL-10 and some other inflammatory mediators in intrauterine tissues and in amniotic fluid differed significantly between the mouse lines after maternal LPS given at 17 dpc. We conclude that SP-A modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.     

Ligand-binding domains in vitellogenin receptors and other LDL-receptor family members share a common ancestral ordering of cysteine-rich repeats

Insect vitellogenin and yolk protein receptors (VgR/YPR) are newly discovered members of the low-density lipoprotein receptor (LDLR) family, which is characterized by a highly conserved arrangement of repetitive modular elements homologous to functionally unrelated proteins. The insect VgR/YPRs are unique in having two clusters of complement-type cysteine-rich (class A) repeats or modules, with five modules in the first cluster and seven in the second cluster, unlike classical LDLRs which have a single seven-module cluster, vertebrate VgRs and very low density lipoprotein receptors (VLDLR) which have a single eight-module cluster, and LDLR-related proteins (LRPs) and megalins which have four clusters of 2–7, 8, 10, and 11 modules. Alignment of clusters across subfamilies by conventional alignment programs is problematic because of the repetitive nature of the component modules which may have undergone rearrangements, duplications, and deletions during evolution. To circumvent this problem, we ``fingerprinted' each class A module in the different clusters by identifying those amino acids that are both relatively conserved and relatively unique within the cluster. Intercluster reciprocal comparisons of fingerprints and aligned sequences allowed us to distinguish four cohorts of modules reflecting shared recent ancestry. All but two of the 57 modules examined could be assigned to one of these four cohorts designated A, B, C, and D. Alignment of clusters based on modular cohorts revealed that all clusters are derived from a single primordial cluster of at least seven modules with a consensus arrangement of CDCADBC. All extant clusters examined are consistent with this consensus, though none matches it perfectly. This analysis also revealed that the eight-module clusters in vertebrate VgRs, insect VgR/YPRs, and LRP/megalins are not directly homologous with one another. Assignment of modules to cohorts permitted us to properly align 32 class A clusters from all four LDLR subfamilies for phylogenetic analysis. The results revealed that smaller one-cluster and two-cluster members of the family did not originate from the breakup of a large two-cluster or four-cluster receptor. Similarly, the LRP/megalins did not arise from the duplication of a two-cluster insect VgR/YPR-like progenitor. Rather, it appears that the multicluster receptors were independently constructed from the same single-cluster ancestor. Received: 16 January 1997 / Accepted: 21 August 1997     

Phylogenetic Analysis of Three Lipocalin-Like Proteins Present in the Milk of Trichosurus vulpecula (Phalangeridae, Marsupialia)

Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents. Received: 28 October 1996 / Accepted: 19 May 1997     

Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo

Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (-/-) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (-/-) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (-/-) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (-/-) animals was decreased by approximately 40%. Instillation of 50 microg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (-/-) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.     

Evolutionary Analysis of the ErbB Receptor and Ligand Families

We have compared all available deduced protein sequences of the ErbB family of receptors and their ligands. Analysis of the aligned sequences of the receptors indicates that there are some differences in the receptors that are specific to invertebrates. In addition, comparison of the vertebrate ErbB receptors suggest that a gene duplication event generated two ancestral receptors, the ErbB3/ErbB4 precursor and the ErbB1/ErbB2 precursor. Subsequent gene duplications of these precursors generated the four receptors present in mammals. Analysis of the sequences for the known ligands of the ErbB receptors suggests that the vertebrate ligands segregate into the ErbB1 ligands and the ErbB3/ErbB4 ligands, paralleling the evolution of the receptors; however, it is difficult to ascertain any correlation between the invertebrate and the vertebrate ligands. Even though ErbB3 is kinase-impaired, there is significant conservation of the kinase domain within the vertebrate lineage (human, rat, and F. rubripes), suggesting some function for this domain other than kinase activity, such as mediating protein–protein interactions that are involved in receptor dimerization and/or activation of the kinase domain of the heterodimerization partner. To date, no ligand for ErbB2 has been identified, and comparison of the extracellular domains of ErbB2 reveals two regions that are not conserved across the mammalian species. These two regions of divergence align with sequences in ErbB1 that have been shown to be proximal to the amino-terminus and to the carboxyl-terminal region, respectively, of bound EGF. Further, one of these regions contains an insertion, relative to the other members of the mammalian ErbB family, which might affect the ligand binding site and provide a structural basis for this receptor's apparent inability to bind ligand independently. Received: 8 September 1999 / Accepted: 17 January 2000     

Molecular Evolution of the Aldo-keto Reductase Gene Superfamily

The aldo-keto reductase enzymes comprise a functionally diverse gene family which catalyze the NADPH-dependant reduction of a variety of carbonyl compounds. The protein sequences of 45 members of this family were aligned and phylogenetic trees were deduced from this alignment using the neighbor-joining and Fitch algorithms. The branching order of these trees indicates that the vertebrate enzymes cluster in three groups, which have a monophyletic origin distinct from the bacterial, plant, and invertebrate enzymes. A high level of conservation was observed between the vertebrate hydroxysteroid dehydrogenase enzymes, prostaglandin F synthase, and ρ-crystallin of Xenopus laevis. We infer from the phylogenetic analysis that prostaglandin F synthase may represent a recent recruit to the eicosanoid biosynthetic pathway from the hydroxysteroid dehydrogenase pathway and furthermore that, in the context of gene recruitment, Xenopus laevisρ-crystallin may represent a shared gene. Received: 26 August 1996 / Accepted: 5 June 1997     

The Molecular Clock Runs at Different Rates Among Closely Related Members of a Gene Family

The serum albumin gene family is composed of four members that have arisen by a series of duplications from a common ancestor. From sequence differences between members of the gene family, we infer that a gene duplication some 580 Myr ago gave rise to the vitamin D–binding protein (DBP) gene and a second lineage, which reduplicated about 295 Myr ago to give the albumin (ALB) gene and a common precursor to α-fetoprotein (AFP) and α-albumin (ALF). This precursor itself duplicated about 250 Myr ago, giving rise to the youngest family members, AFP and ALF. It should be possible to correlate these dates with the phylogenetic distribution of members of the gene family among different species. All four genes are found in mammals, but AFP and ALF are not found in amphibia, which diverged from reptiles about 360 Myr ago, before the divergence of the AFP-ALF progenitor from albumin. Although individual family members display an approximate clock-like evolution, there are significant deviations—the rates of divergence for AFP differ by a factor of 7, the rates for ALB differ by a factor of 2.1. Since the progenitor of this gene family itself arose by triplication of a smaller gene, the rates of evolution of individual domains were also calculated and were shown to vary within and between family members. The great variation in the rates of the molecular clock raises questions concerning whether it can be used to infer evolutionary time from contemporary sequence differences. Received: 28 February 1995 / Accepted: 6 October 1997     

Phylogenies of Developmentally Important Proteins Do Not Support the Hypothesis of Two Rounds of Genome Duplication Early in Vertebrate History

It has been proposed that two rounds of duplication of the entire genome (polyploidization) occurred early in vertebrate history (the 2R hypothesis); and the observation that certain gene families important in regulating development have four members in vertebrates, as opposed to one in Drosophila, has been adduced as evidence in support of this hypothesis. However, such a pattern of relationship can be taken as support of the 2R hypothesis only if (1) the four vertebrate genes can be shown to have diverged after the origin of vertebrates, and (2) the phylogeny of the four vertebrate genes (A–D) exhibits a topology of the form (AB) (CD), rather than (A) (BCD). In order to test the 2R hypothesis, I constructed phylogenies for nine protein families important in development. Only one showed a topology of the form (AB) (CD), and that received weak statistical support. In contrast, four phylogenies showed topologies of the form (A) (BCD) with statistically significant support. Furthermore, in two cases there was significant support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes: in one case there was significant support for duplication of the vertebrate genes at least prior to the divergence of vertebrates and urochordates, and in one case there was weak support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes. Taken together with other recently published phylogenies of developmentally important genes, these results provide strong evidence against the 2R hypothesis. Received: 22 December 1997 / Accepted: 5 October 1998     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

Monophyly of Lampreys and Hagfishes Supported by Nuclear DNA–Coded Genes

The phylogenetic position of hagfishes in vertebrate evolution is currently controversial. The 18S and 28S rRNA trees support the monophyly of hagfishes and lampreys. In contrast, the mitochondrial DNAs suggest the close association of lampreys and gnathostomes. To clarify this controversial issue, we have conducted cloning and sequencing of the four nuclear DNA–coded single-copy genes encoding the triose phosphate isomerase, calreticulin, and the largest subunit of RNA polymerase II and III. Based on these proteins, together with the Mn superoxide dismutase for which hagfish and lamprey sequences are available in database, phylogenetic trees have been inferred by the maximum likelihood (ML) method of protein phylogeny. It was shown that all the five proteins prefer the monophyletic tree of cyclostomes, and the total log-likelihood of the five proteins significantly supports the cyclostome monophyly at the level of ±1 SE. The ML trees of aldolase family comprising three nonallelic isoforms and the complement component group comprising C3, C4, and C5, both of which diverged during vertebrate evolution by gene duplications, also suggest the cyclostome monophyly. Received: 28 April 1999 / Accepted: 30 June 1999     

Surfactant Protein A in Cystic Fibrosis: Supratrimeric Structure and Pulmonary Outcome

Background

The state of oligomerization of surfactant associated protein-A (SP-A) monomers differs between individuals. This likely affects SP-A’s functional properties and could thereby influence clinical status in patients with lung diseases. In this study we focus on SP-A structure in cystic fibrosis (CF) compared to both healthy subjects and disease controls.

Methods

SP-A composition and function were assessed in both bronchoalveolar lavage (BAL) fluid and serum of 46 CF patients with mild disease, 25 patients with chronic bronchitis and 22 healthy subjects by gel chromatography and a functional agglutination assay. Relation of SP-A agglutination ability to disease severity of the subjects was explored.

Results

SP-A was present in seven major oligomeric forms with the majority of SP-A being structurally organized as complex oligomeric forms. More complex oligomeric forms were associated with better SP-A function with regard to its agglutination ability. These forms were more frequently observed in BAL than in serum, but there were no differences between disease groups. In CF patients, more complex forms of SP-A were associated with better lung function.

Conclusions

Organizational structure of SP-A affects its functional activity and is linked to disease severity in CF.     

The Path from the RNA World

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA → RNP → protein, the first proteins in the ``breakthrough organism' (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the ``introns-first' theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a ``eukaryotic-like' genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome → eukaryotic-like DNA genome → prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome → eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA → RNP → protein. A likely molecular mechanism, ``plasmid transfer,' is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history. Received: 14 January 1997 / Accepted: 19 May 1997     

Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by β-strands 6 and 7 of the crystal structures of bovine RIα and rat RIIβ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–[PV]–R–[ANQV]–A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects α-helix B′ and β strand 7. Received: 2 November 2000/Accepted: 14 June 2001     

The BC200 RNA Gene and Its Neural Expression Are Conserved in Anthropoidea (Primates)

The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea. The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized by a significantly higher substitution rate in the examined 5′ flanking region than in the BC200 RNA coding region itself. Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5′ flanking region (∼400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that the gene encoding BC200 RNA was created at least 35–55 million years ago and its presence, mode of expression, and association with protein(s) as an RNP are under selective pressure. Received: 1 December 1997 / Accepted: 3 June 1998     

The MADS-box floral homeotic gene lineages predate the origin of seed plants: Phylogenetic and molecular clock estimates

Flower development in angiosperms is controlled in part by floral homeotic genes, many of which are members of the plant MADS-box regulatory gene family. The evolutionary history of these developmental genes was reconstructed using 74 loci from 15 dicot, three monocot, and one conifer species. Molecular clock estimates suggest that the different floral homeotic gene lineages began to diverge from one another about 450–500 mya, around the time of the origin of land plants themselves. Received: 31 January 1997 / Accepted: 9 April 1997     

Isolation of Heavy Endosomes from Dog Proximal Tubules in Suspension

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42–44 kDa and 20–24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of β-COP, β-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as ``heavy endosomes' (buoyant density D= 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules. Received: 13 July 1995/Revised: 8 May 1996