On the phylogenetic position of the pre-Neandertal specimen from Reilingen, Germany

This paper describes the morphology and phylogenetic significance of a well-preserved fossil human cranium found near Reilingen, Germany in 1978. The specimen consists of two complete parietals, most of the right temporal, and 70% of the occipital. The specimen displays several features characteristic of early "archaic" Homo sapiens: maximum breadth at the supramastoid crests, roughly pentagonal occipital view, an angular torus, superoinferiorly elongate/ anteroposterior shortened squamous temporal, strong mastoid, and separation of the tympanic and mastoid temporal portions. Other features align the Reilingen cranium with the "classic" Neandertals: developed suprainiac fossa, bilaterally protuberant occipital torus, strong juxtamastoid eminence, a highly convex occipital plane, and lambdoid flattening. This morphology and our cephalometric analysis support the assignment of this specimen to a Stage 2 "pre-Neandertal" group (e.g., Steinheim, Swanscombe, and Atapuerca [SH site]). Following an "Accretion Model" of European hominid evolution, Stage 2 succeeds Stage 1 European "early-pre-Neandertals" (e.g., Mauer and Petralona), and precedes both the Saale-Eem Stage 3 "early Neandertals" (e.g., Biache 1), which present greater platycephaly, a reduced mastoid process, an enlarged juxtamastoid eminence, and true "en-bombe" (parieto-occipital form), and Weichsel Stage 4 "Neandertals" (sensu stricto, i.e., "classic" Neandertals).     

Sequence analysis and immunofluorescence study of alpha- and beta-tubulins in Reticulomyxa filosa: implications of the high degree of beta2-tubulin divergence

We have cloned and sequenced 2 alpha- and 2 beta-tubulin isoforms from the giant freshwater amoeba Reticulomyxa filosa. The microtubules of this organism exhibit some unusual properties, including the highest rates of assembly and disassembly known and the inability to be stabilized by taxol. The cloned alpha-tubulins show a high degree of identity when compared to an alpha-tubulin consensus sequence. The beta-tubulins, however, are more divergent, the beta2-tubulin being the most unusual beta-tubulin found so far. The deduced amino acid sequence of beta2 shows 55% identity to a beta-tubulin consensus sequence. It also features 51 unique exchanges which cluster in the C-terminal half of the molecule. Several unique exchanges and two insertions occur in regions adjacent to, or directly implicated in, conserved beta-tubulin functions. A phylogenetic analysis places the beta-tubulins of R. filosa in the vicinity of beta-tubulins from fungi and slime molds. Monoclonal and polyclonal antibodies raised against R. filosa tubulins show that the electrophoretic mobility of alpha- and beta-tubulins is reversed with respect to tubulins from most other sources. Immunofluorescence experiments reveal a ubiquitous distribution of both beta-tubulins in the amoebal network. Our observations suggest possible links between the aberrant primary structure of the beta2-tubulin and the unusual properties of R. filosa microtubules.     

Molecular characterization and phylogenetic distribution of the streptococcal superantigen gene (ssa) from Streptococcus pyogenes

A striking increase in the frequency and severity of invasive infections caused by Streptococcus pyogenes has occurred in recent years. Among these diseases is streptococcal toxic-shock-like syndrome (TSLS), a condition characterized by fulminant soft-tissue destruction and multiorgan failure. Streptococcal superantigen (SSA), a superantigen isolated from a TSLS-inducing, serotype M3 S. pyogenes strain, has recently been identified. We here describe the cloning, sequencing, and phylogenetic distribution of the SSA structural gene. The 783-bp open reading frame encodes a predicted 260-amino-acid protein that is similar in size to several other bacterial superantigens. The deduced sequence of the mature protein is 60.2% identical to that of staphylococcal enterotoxin B but only 49% identical to that of streptococcal pyrogenic exotoxin A. Southern blot and PCR analysis of 138 group A streptococcal strains representing 65 M protein serotypes and 15 nontypeable isolates identified ssa in 68 strains from 10 distinct clonal lineages. All ssa-positive clones expressed SSA. Of the two clones associated with TSLS, the ET 2-M3 lineage, but not the ET 1-M1 lineage, carried the SSA gene. Further analysis of the ET 2-M3 lineage found evidence for temporal variation in ssa association. Contemporary ET 2-M3 disease isolates had ssa, but two older isolates of this clone recovered in 1910 and 1920 lacked the gene. The clonal and temporal distribution patterns of ssa suggest a relatively recent acquisition of this superantigen-encoding gene by the ET 2-M3 lineage, perhaps by horizontal transfer and recombination.     

The phylogenetic status of phyllomedusine frogs (Hylidae) as evidenced from immunological studies of their serum albumins

Based on immunological comparisons of the serum albumins of phyllomedusine frogs with both hyline and bufonid species, it is suggested that phyllomedusine frogs be erected to familial status within the superfamily Bufonoidea.     

Acute phase proteins: alpha-1-acid glycoprotein (AGP) and alpha-1 antichymotrypsin (ACT) in serum of patients with cerebral ischemic stroke

BACKGROUND: Because of methods required for obtaining isolated left ventricular myocytes, evaluation of the contractile function of isolated left ventricular myocytes in normal human patients has been limited. Accordingly, the goal of the present study was to develop a means to isolate human left ventricular myocytes from small myocardial biopsy specimens collected from patients undergoing elective coronary artery bypass operations and to characterize indices of myocyte contractile performance. METHODS: Myocardial biopsy specimens were obtained from the anterior left ventricular free wall of 22 patients undergoing coronary artery bypass operations. Myocytes were isolated from these myocardial samples by means of a stepwise enzymatic digestion method and micro-trituration techniques. Isolated left ventricular myocyte contractile function was assessed by computer-assisted high-speed videomicroscopy under basal conditions and in response to beta-adrenergic receptor stimulation with isoproterenol. RESULTS: A total of 804 viable left ventricular myocytes were successfully examined from all of the myocardial biopsy specimens with an average of 37+/-4 myocytes per patient. All myocytes contracted homogeneously at a field stimulation of 1 Hz with an average percent shortening of 3.7%+/-0.1% and shortening velocity of 51.3+/-1.3 microm/s. After beta-adrenergic receptor stimulation with isoproterenol, percent shortening and shortening velocity increased 149% and 118% above baseline, respectively (P < .05). CONCLUSION: The unique results of the present study demonstrated that a high yield of myocytes could be obtained from human left ventricular biopsy specimens taken during cardiac operations. These myocytes exhibited stable contractile performance and maintained the capacity to respond to an inotropic stimulus. The methods described herein provide a basis by which future studies could investigate intrinsic and extrinsic influences on left ventricular myocyte contractility in human beings.     

Nuclear gene trees and the phylogenetic relationships of the mangabeys (Primates: Papionini)

Phylogenetic relationships of mangabeys within the Old World monkey tribe Papionini are inferred from analyses of nuclear DNA sequences from five unlinked loci. The following conclusions are strongly supported, based on congruence among trees derived for the five separate gene regions: (1) mangabeys are polyphyletic within the Papionini; (2) Cercocebus is the sister taxon to the genus Mandrillus; and (3) Lophocebus belongs to a clade with Papio and Theropithecus, with Papio as its most likely sister taxon. Morphologically based phylogenies positing mangabey monophyly were evaluated by mapping the sequences for each locus on these trees. The data seem to fit these trees poorly in both maximum-parsimony and likelihood analyses. Incongruence among nuclear gene trees occurred in the interrelationships among Lophocebus, Papio, and Theropithecus. Several factors that may account for this incongruence are discussed, including sampling error, random lineage sorting, and introgression.     

Determination of the sialylation level and of the ratio alpha-(2-->3)/alpha-(2-->6) sialyl linkages of N-glycans by methylation and GC/MS analysis

A methodology for the determination of the sialylation pattern of N-glycans, extent of sialylation and the ratio between alpha-(2-->3) and alpha-(2-->6) sialyl linkages, is presented based on the labelling of the C-3 and C-6 hydroxyl groups of Gal residues obtained after permethylation, saponification, selective desialylation of sialylated oligosaccharides and methanolysis. Deuteromethylation and GC/MS analysis of Gal derivatives allow to determine the sialylation level of glycans. O-Ethyl ether labelling followed by GC analysis of the resulting Gal derivatives allows to obtain the ratio between alpha-(2-->3) and alpha-(2-->6) sialyl linkages. The method was applied to LNT (LcOse4: beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)-beta-D- Galp-(1-->4)-D-Glcp), LSTa (IV3NeuAcLcOse4: alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D- GlcpNac-(1-->3-beta-D-Galp-(1-->4)-D-Glcp), LSTc (IV6NeuAcn LcOse4: alpha-Neup5Ac-(2-->6)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)- beta-D-Galp-(1-->4)-D-Glcp) and a bisialylated biantennary N-glycan in which sialic acid is bound to Gal residues via an alpha-(2-->6) linkage. Using this method, it was found that 92.8% of N-glycans in bovine fetuin is sialylated and that the ratio of alpha-(2-->6) versus alpha-(2-->3) sialyl linkages was 31:19.     

Genetic and phylogenetic divergence of feline immunodeficiency virus in the puma (Puma concolor)

Feline immunodeficiency virus (FIV) is a lentivirus which causes an AIDS-like disease in domestic cats (Felis catus). A number of other felid species, including the puma (Puma concolor), carry a virus closely related to domestic cat FIV. Serological testing revealed the presence of antibodies to FIV in 22% of 434 samples from throughout the geographic range of the puma. FIV-Pco pol gene sequences isolated from pumas revealed extensive sequence diversity, greater than has been documented in the domestic cat. The puma sequences formed two highly divergent groups, analogous to the clades which have been defined for domestic cat and lion (Panthera leo) FIV. The puma clade A was made up of samples from Florida and California, whereas clade B consisted of samples from other parts of North America, Central America, and Brazil. The difference between these two groups was as great as that reported among three lion FIV clades. Within puma clades, sequence variation is large, comparable to between-clade differences seen for domestic cat clades, allowing recognition of 15 phylogenetic lineages (subclades) among puma FIV-Pco. Large sequence divergence among isolates, nearly complete species monophyly, and widespread geographic distribution suggest that FIV-Pco has evolved within the puma species for a long period. The sequence data provided evidence for vertical transmission of FIV-Pco from mothers to their kittens, for coinfection of individuals by two different viral strains, and for cross-species transmission of FIV from a domestic cat to a puma. These factors may all be important for understanding the epidemiology and natural history of FIV in the puma.     

Reaction of the cardio-respiratory-system following acetylcholine inhalation and the dependency from the body position on anaesthetised dogs (author's transl)

The bronchomotoric reaction following ACH-aerosol in low concentrations is dependend on the position of the body on dogs. Up till one maximal reaction the increase of the airway resistance is always stronger in the lateral position. Nevertheless the breathing minute volume is constant. Concentration reaction curves will be shown. Arterial blood gases, heart rate, and pressure in the arteria femoralis were also measured. The different strongness of the reaction following ACH-inhalation will be caused by the predominantely unilateral ventilation of the animals in the lateral position.     

The cultivation of Fossaria cubensis (Pfeiffer) (Pulmonata: Lymnaeidae), the intermediate host of Fasciola hepatica (Linnaeus) in Cuba

A method for the culture of Fossaria cubensis (Pfeiffer), the major intermediate host of fascioliasis in Cuba, was assessed. For this purpose, an algae-bearing plate was designed based on a culture of Cyanophyceae algae from Lyngbya, Leotolyngbya, Phormidium, and Schimidleinema on Petri plates 12 cm in diameter with mud substrate and high levels of nitrate and calcium carbonate. Fifteen days after incubation with continuous light, these plates were used as aquaria for mollusc breeding. Under such conditions the generational time is reduced and the growth and reproduction of Fossaria cubensis which allow for their mass production aimed at the research of fascioliasis is guaranteed.     

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.     

The role of host (endogenous) T cells in chronic graft-versus-host autoimmune disease

Chronic graft-vs-host (cGVH) disease induced by the transfer of Ia-incompatible spleen cells from one normal mouse strain (such as B6.C-H2(bm12)/KhEg (bm12)) to another (such as C57BL/6) causes an autoimmune syndrome resembling systemic lupus erythematosus (SLE). The role of host-derived T cells in this response is not obvious. Previous reports suggested that host T cells might serve to down-regulate the autoimmune syndrome. To address this issue more definitively, we used CD4 knockout (KO) or CD8KO C57BL/6 (B6) mice as recipients in the bm12-->C57B6 cGVH model. CD4KO B6 mice injected with allogeneic bm12 spleen cells (bm12-->CD4KO group) showed no evidence of cGVH disease. They made no detectable autoantibodies, including anti-chromatin, anti-dsDNA, anti-ssDNA, and rheumatoid factor. They survived at least 20 wks after induction of cGVH disease; and they did not develop nephritis, based on the absence of detectable levels of proteinuria and normal renal histology at the time of sacrifice. By contrast, CD8KO B6 mice (bm12-->CD8KO group) and normal B6 mice (bm12-->B6 group) injected with bm12 spleen cells generally showed similar levels of mortality, nephritis, and autoantibodies, although the autoantibody titers declined somewhat after week 8 in the bm12-->CD8KO group. Control groups of recipients injected with B6 spleen cells showed no induction of autoantibodies. A surprising finding, however, was that the B6-->CD8KO group developed severe histologic glomerulonephritis in the absence of autoantibodies and with decreased immune deposits. These results indicate that endogenous (host) CD4+ T cells play an essential role in the cGVH autoimmune syndrome.     

The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA.     

Synthesis of optically active alpha-(p-chlorophenyl)pyridylmethanols with plant cell cultures

We report here on the mapping of a cDNA encoding for human cysteine-rich heart protein (HCRHP), a counterpart of the murine cysteine-rich intestinal protein CRIP. By somatic cell hybrid analysis and radiation hybrid mapping, we have located the gene CRIP1 (HGMW-approved symbol) on the subcentromeric region of the q arm of human chromosome 7, flanking a deletion associated with Williams syndrome.     

Biochemical and phylogenetic characterization of the dUTPase from the archaeal virus SIRV

The derived amino acid sequence from a 474-base pair open reading frame in the genome of the Sulfolobus islandicus rod-shaped virus SIRV shows striking similarity to bacterial dCTP deaminases and to dUTPases from eukaryotes, bacteria, Poxviridae, and Retroviridae. The putative gene was expressed in Escherichia coli, and dUTPase activity of the recombinant enzyme was demonstrated by hydrolysis of dUTP to dUMP. Deamination of dCTP by the enzyme was not detected. Phylogenetic analysis based on amino acid sequences of the characterized enzyme and its homologues showed that the dUTPase-encoding dut genes and the dCTP deaminase-encoding dcd genes constitute a paralogous gene family. This report is the first identification and functional characterization of an archaeal dUTPase and the first phylogeny derived for the dcd-dut gene family.     

The technique of anesthesia for the prevention and treatment of gaseous embolism in the neurosurgical sitting position (author's transl)

The sitting position in neurosurgery gives always three problems for the anaesthesist: --maintain the cerebral perfusion pressure, --not to aggravate intracranial hypertension, --detect gaseous embolism. The clinical signs of air embolism often look like the awakening of the patient. That is why it is necessary to use specific methods to detect them: Doppler, Swan-Ganz catheter, and/or continuous CO2 recording of expired air by capnograph. Among the various methods to prevent air embolism we have used controlled respiration with a positive and expiratory pressure (P.E.E.P.). In our series (45 operations) gas embolism occurred in 13% of the patients operated upon without positive and expiratory pressure, and 0% with P.E.E.P.     

Molecular phylogenetic information on the identity of the closest living relative(s) of land vertebrates

The phylogenetic position of tetrapods relative to the other two living sarcopterygian lineages (lungfishes and the coelacanth) has been subject to debate for many decades, yet remains unresolved. There are three possible alternatives for the phylogenetic relationships among these three living lineages of sarcopterygians, i.e., lungfish as living sister group of tetrapods, the coelacanth as closest living relative of tetrapods, and lungfish and coelacanth equally closely related to tetrapods. To resolve this important evolutionary question several molecular data sets have been collected in recent years, the largest being the almost complete 28S rRNA gene sequences (about 3500 bp) and the complete mitochondrial genomes of the coelacanth and a lungfish (about 16,500 bp each). Phylogenetic analyses of several molecular data sets had not provided unequivocal support for any of the three hypotheses. However, a lungfish + tetrapod or a lungfish + coelacanth clade were predominantly favored over a coelacanth + tetrapod grouping when the entire mitochondrial genomes alone or in combination with the nuclear 28S rRNA gene data were analyzed with maximum parsimony, neighbor-joining, and maximum likelihood phylogenetic methods. Also, current paleontological and morphological data seem to concur with these molecular results. Therefore the currently available molecular data seems to rule out a coelacanth + tetrapod relationship, the traditional textbook hypothesis. These tentative molecular phylogenetic results point to the inherent difficulty in resolving relationships among lineages which apparently originated in rapid succession during the Devonian.     

Recovering phylogenetic signal from DNA sequences: relationships within the corvine assemblage (class aves) as inferred from complete sequences of the mitochondrial DNA cytochrome-b gene

Phylogenetic analysis of cytochrome-b sequences and cranial osteological characters for nine genera of corvine passerine birds supports the hypothesis that the two major groups of birds of paradise, the manucodines and paradisaeinines, constitute a monophyletic group and that their postulated sister group is the Corvidae (crows, jays, and allies). The data are also consistent with the hypothesis that the bowerbirds are not closely related to the birds of paradise but instead lie near the base of the corvine assemblage. The corvine radiation exemplifies a case of multiple star phylogenies embedded within a major clade, with the branching pattern characterized by very short internodal divergence times. Such histories are difficult to resolve no matter what type of data is employed, because little change accumulates between branching events. With respect to sequence data, reconstructed tree topologies are sensitive to the choice of outgroup and to the method of analysis (e.g., transversion vs. global parsimony). In such cases, assessing the "reliability" of a best-fit or most-parsimonious tree inferred from any particular data set becomes problematic. Statistical tests of tree topologies that depend on random sampling of characters will generally be inconclusive in that all cladistic components will tend to be poorly supported because relatively few character-state changes will be recorded between branching events. It is suggested, on the other hand, that congruence in cladistic signal across different data sets may be a potentially more useful method for evaluating the reliability of the signal of any one data set. Resolution of star phylogenies will probably be possible only if DNA sequence and morphological characters are combined in a single analysis.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.