Phylogenetic systematic evaluation of a classification of the Zoogonidae Odhner, 1902 (Cercomeria: Trematoda: Digenea: Plagiorchiiformes)

A recent systematic study of the digenean family Zoogonidae presented a series of cladograms, which are the product of phylogenetic systematic, or cladistic, analysis. However, one of the two subfamilies and nine of the 21 genera recognised in that study lacked putative synapomorphies, a requirement for phylogenetic systematic studies. This study presents a re-analysis of the database for the zoogonids, based on rigorous application of phylogenetic systematic methods. A new phylogenetic tree is presented, which better fits the original data than the published tree (with a consistency index of 52.3% vs. 46.3%). Four subfamilies, three monophyletic and one of uncertain status, and 10 genera could be recognised phylogenetically. This would affect the nomenclatorial status of one-third (26) of the species in the family. However, it is recommended that another analysis, based on more characters, be carried out before nomenclatorial changes are proposed.     

Unusual responses of proboscis muscles ofBusycon canaliculatum to some calcium antagonist agents

Summary K- and ACh-induced responses of the radular sac, odontophore retractor, and radular retractor muscles ofBusycon canaliculatum were found to be strongly dependent upon [Ca]₀. Diltiazem had strong positive inotropic and chronotropic actions on fast twitch activity in the odontophore retractor and radular protractor muscles. K-induced tonic force in these muscles was partly inhibited by diltiazem but only at very high concentrations. ACh responses in all muscles were eliminated by diltiazem. Nifedipine enhanced fast twitches and tonic force in response to high K, and induced persistent spontaneous fast twitch discharges. Nifedipine inhibited ACh-induced tonic force, but induced rhythmic bursts of fast twitches persisting long after nifedipine washout. Verapamil strongly inhibited K- and ACh-induced tonic force in all three muscles at high concentration, but stimulated fast twitch responses and converted ACh contractures into fast twitch activity. Sucrose gap studies showed that nifedipine and diltiazem reduced K- and ACh-induced tension and depolarization. Paradoxically, verapamil reduced K- and ACh-induced tension but significantly enhanced their induced depolarizations. Diltiazem, nifedipine and verapamil did not act like slow Ca channel antagonists in these muscles. This may reflect differences in channel structure between molluscs and mammals, or differences in the cellular calcium release pathways operated by such channels in molluscan and mammalian muscle. These Ca-ant-agonists appeared to act as agonists of fast twitch activity in these muscles and antagonists of the ACh-induced calcium release pathway for tonic force development.     

Differing T-cell requirements for recombinant retrovirus vaccines.

Friend murine leukemia virus is a retrovirus complex that induces rapid erythroleukemia and immunosuppression in susceptible strains of adult mice. Using this model, we directly examined the T-cell subsets required for a protective retrovirus vaccine. Paradoxically, recovery in mice immunized with a chimeric envelope containing only T-helper (TH) and B-cell epitopes was dependent on CD8+ T cells as well as CD4+ T cells despite the fact that the vaccine contained no CD8+ cytolytic T-lymphocyte (CTL) epitopes. However, the requirement for CD8+ T cells was overcome by inclusion of additional TH and B-cell epitopes in the immunizing protein. These additional epitopes primed for more rapid production of virus-neutralizing antibody which appeared to limit virus spread sufficiently to protect even in the absence of CD8+ T cells. Inclusion of an immunodominant CTL epitope in the vaccine was not sufficient to overcome dependence on CD4+ T cells. These data suggest that TH priming is more critical for retrovirus immunity than CTL priming.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): six unique arylsulfatase B gene alleles causing variable disease phenotypes.

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase B (ASB), also known as N-acetylgalactosamine-4-sulfatase. Multiple clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of the human ASB gene facilitated the analysis of molecular defects underlying the different phenotypes. Conditions for PCR amplification of the entire open reading frame from genomic DNA and for subsequent direct automated DNA sequencing of the resulting DNA fragments were established. Besides two polymorphisms described elsewhere that cause methionine-for-valine substitutions in the arylsulfatase B gene, six new mutations in six patients were detected: four point mutations resulting in amino acid substitutions, a 1-bp deletion, and a 1-bp insertion. The point mutations were two G-to-A and two T-to-C transitions. The G-to-A transitions cause an arginine-for-glycine substitution at residue 144 in a homoallelic patient with a severe disease phenotype and a tyrosine-for-cysteine substitution at residue 521 in a potentially heteroallelic patient with the severe form of the disease. The T-to-C transitions cause an arginine-for-cysteine substitution at amino acid residue 192 in a homoallelic patient with mild symptoms and a proline-for-leucine substitution at amino acid 321 in a homoallelic patient with the intermediate form. The insertion between nucleotides T1284 and G1285 resulted in a loss of the 100 C-terminal amino acids of the wild-type protein and in the deletion of nucleotide C1577 in a 39-amino-acid C-terminal extension of the ASB polypeptide. Both mutations were detected in homoallelic patients with the severe form of the disease. Expression of mutant cDNAs encoding the four amino acid substitutions and the deletion resulted in severe reduction of both ASB protein levels and arylsulfatase enzyme activity in comparison with a wild-type control. The six mutations described in the present study were unique among 25 unrelated mucopolysaccharidosis VI patients, suggesting a broad molecular heterogeneity of the Maroteaux-Lamy syndrome.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Development of physical forms of unintegrated retroviral DNA in mouse spinal cord tissue during ts1-induced spongiform encephalomyelopathy: elevated levels of a novel single-stranded form in paralyzed mice.

ts1 is a murine leukemia virus that causes rapidly evolving hindlimb paralysis in susceptible strains of mice. Following perinatal infection, three physical forms of unintegrated viral DNA were detected in the spinal cord by Southern blot hybridization. Linear and supercoiled closed-circle viral double-stranded DNAs were detected in both the central nervous system and non-central nervous system tissues. An elevated level of a novel minus-sense single-stranded form of viral DNA, which had a very high mobility in agarose gels, was correlated with the onset of symptoms of paralysis. As the severity of paralysis progressed, the level of this single-stranded form increased rapidly, with the highest level in the spinal cords of moribund mice. Since the virulence of a number of cytopathic retroviruses has been associated with the presence of increased amounts of unintegrated viral DNA in the tissues of the infected hosts, this novel form of highly mobile unintegrated single-stranded DNA may have a role in the neuropathogenesis of ts1.