Phylogenetic analysis of the Rhabdocoela (Platyhelminthes) with emphasis on the Neodermata and relatives

Phylogenetic systematic analysis of 24 taxa representing the rhabdocoel platyhelminths, based on a suite of 89 morphological characters, produced two equally parsimonious trees, 181 steps long, with a consistency index (CI) of 0.69 and a rescaled consistency index (RCI) of 0.56, differing only with respect to that portion of the tree containing Umagillidae, Acholadidae, Graffillinae, Pseudograffillinae, Pterastericolidae and Hypoblepharinidae. Our results accommodate all previously proposed sister taxa to the Neodermata in a single clade in which ((Dalyelliidae + Temnocephalida) Typhloplanidae) is the sister group of ((Fecampiidae +  Urastoma ) ( Udonella ((Aspidogastrea + Digenea) (Monogenea (Gyrocotylidea (Amphilinidea + Eucestoda)))))). Bootstrap and jackknife analyses indicate that the groupings of ((Dalyelliidae + Temnocephalida) Typhloplanidae) and of ((Fecampiidae +  Urastoma ) ( Udonella ((Aspidogastrea + Digenea) (Monogenea (Gyrocotylidea (Amphilinidea + Eucestoda)))))) are highly robust, with the latter clade having a CI of 90% and RCI of 82%. Disagreements among previous analyses of these taxa have been due to the influence of missing data for critical characters in key taxa and differences in the taxa analysed, rather than any inherent weakness in the morphological data. Non-phylogenetic systematic approaches to homology assessment and misconceptions regarding phylogenetic systematic methodology are discussed. Recent analyses combining sequence data with a subset of approximately 60% of the morphological characters should be re-assessed using the entire morphological database. Even if Udonella is a monogenean, it is most parsimonious to suggest that the common ancestor of the Neodermata had a vertebrate–arthropod two-host life cycle.     

The metabolism of synthetic leukotriene B4 in synovial fluid and whole human blood

The metabolism in vitro of synthetic leukotriene B4 (LTB4) in synovial fluid from rheumatoid arthritis and osteoarthritis patients and in whole blood from these same patient groups and from normal volunteers has been studied. A linear relationship existed between a plot of the time of incubation of samples with LTB4 and the percentage of the initial concentration of LTB4 at each time point. The slope of this line, the rate constant for metabolism, has been used to compare different samples. LTB4 was metabolised more rapidly in the synovial fluid of rheumatoid arthritis patients than osteoarthritis patients. Furthermore, LTB4 was metabolised more rapidly in the blood of rheumatoid arthritis patients than either osteoarthritis patients or normal volunteers. These differences in metabolism correlate with the polymorphonuclear leukocyte (PMN) and albumin content of samples. It is suggested that binding of LTB4 to albumin in vivo will in part determine the available concentration of LTB4 in inflammatory lesions.     

Drug modification of protein thiophosphorylation in permeabilized chromaffin cells

Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5′-O-(3-thiotriphosphate)[³⁵S] results in ³⁵S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permeabilized cells were treated with various drugs known to influence cell functions, more specifically chromaffin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function.

Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or ³⁵S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation.

Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4′diisothiocyanatostilbene-2,2′disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thiophosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.     

Growth measurements of terrestrial microbial species by a continuous-flow technique

A continuous nutrient flow system has been developed to measure microbial activity in soil with various concentrations of added substrate. The system consists of a thin soil layer through which substrate was added continuously over periods up to 4.5 days. Substrate utilization was determined by effluent analysis. Respiration was measured manually by injecting a sample into a gas chromatograph or automatically by coupling the growth chamber to a computer-controlled gas sampling valve. This permitted respiratory CO₂ to be measured by the gas chromatograph at intervals selected by the investigator. Software controlling the valve and gas chromatograph not only automated gas phase sampling, but also provided a scan of CO₂ evolution and a preliminary data summary. This included the date and time of sample, peak height, and percent CO₂ in the gas phase. Data for growth on glucose using a microbial population native to a California annual grassland soil demonstrated that the direct cell count and respiratory techniques for biomass estimation give comparable results. This procedure provides the potential for detailed analyses of substrate utilization in studies of the growth and maintenance of soil microorganisms.     

Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit Mr values.

Initial purification of N-acetylgalactosamine-4-sulphate sulphatase from human liver homogenates containing approx. 1 mg of enzyme in 26 g of soluble proteins was achieved by a six-column chromatography procedure and yielded approx. 40 micrograms of a single major protein species. Enzyme thus prepared was used to produce N-acetylgalactosamine-4-sulphate sulphatase-specific monoclonal antibodies. The use of a monoclonal antibody linked to a solid support facilitated the purification of approx. 0.5 mg of N-acetylgalactosamine-4-sulphate sulphatase from a similar liver homogenate. Moreover the enzyme isolated contained a single protein species, shown by SDS/polyacrylamide-gel electrophoresis to have an Mr of 57,000, which dissociated into subunits of Mr 43,000 and 13,000 in the presence of reducing agents. Essentially identical enzyme preparations were isolated from homogenates of human kidney and lung and from concentrated human urine. The native protein Mr of enzyme from human liver and kidney was assessed by gel-permeation chromatography to be 43,000 on Ultrogel AcA and Bio-Gel P-150. The liver N-acetylgalactosamine-4-sulphate sulphatase was shown to have pH optima of approx. 4 and 5.5 with the oligosaccharide substrate (GalNAc4S-GlcA-GalitolNAc4S) and fluorogenic substrate (methylumbelliferyl sulphate) respectively. Km values of 60 microM and 4 mM and Vmax. values of 2 and 20 mumol/min per mg were determined with the oligosaccharide and fluorogenic substrates respectively.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP/ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent Km value for MgATP2- (at saturating creatine) gives a value of 36 microM when MiMi-CK is coupled to oxidative phosphorylation. This Km value is threefold lower than the Km for enzyme bound to mitoplasts or free in solution. The nucleotide translocase Km value for ADP decreases from 20 to 10 microM in the presence of 50 mM creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased Km values. The observed Km differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the Km values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395-14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent Km values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.     

Nucleotide sequence of the DdeI restriction-modification system and characterization of the methylase protein.

The DdeI restriction-modification system was previously cloned and has been maintained in E. coli on two separate and compatible plasmids (1). The nucleotide sequence of the endonuclease and methylase genes has now been determined; it predicts proteins of 240 amino acids, Mr = 27,808, and 415 amino acids, Mr = 47,081, respectively. Inspection of the DNA sequence shows that the 3' end of the methylase gene had been deleted during cloning. The clone containing the complete methylase gene was made and compared to that containing the truncated gene; only clones containing the truncated form support the endonuclease gene in E. coli. Bal-31 deletion studies show that methylase expression in the Dde clones is also dependent upon orientation of the gene with respect to pBR322. The truncated and complete forms of the methylase protein were purified and compared; the truncated form appears to be more stable and active in vitro. Finally, comparison of the deduced amino acid sequence of M. DdeI with that of other known cytosine methylases shows significant regions of homology.