The male-sterility polymorphism of Silene vulgaris: analysis of genetic dat from two populations and comparison with Thymus vulgaris

Results are given of genetic studies of male sterility using plants from two natural populations from Sussex, England. Both populations have substantial frequencies of females, approximately 0.25 in population 1 and 0.60 in population 3. As in the few other gynodioecious populations studied in detail, many genetic factors are present. In population 1, there are at least two, and more likely three, different cytoplasmic types, one of which appears to produce male sterility in progeny from any hermaphrodite pollen donor; in other words restorer alleles for this cytoplasm are rare or absent from the population. The other two populations can be carried in hermaphrodites that have the dominant restorers. In population 1, there are also probably three restorer loci with complementary recessive male-sterility alleles, as well as a locus with duplicate action, which cannot produce male sterility unless the plant is also homozygous for the recessive allele at another locus. The results from population 3 are quite similar, though there was no evidence in this population for an unrestored sterility cytoplasm. A similar joint nucleocytoplasmic model with multiple restorers fits data from Thymus vulgaris.     

Phylogenetic analysis of parthenogenesis-inducing Wolbachia in the genus Aphytis (Hymenoptera: Aphelinidae)

Well differentiated squamous cell carcinoma of the nasal septum developed in a 55-year old man with Wegener's granulomatosis. It is suggested that the malignancy was induced by immunosuppressive state from an increased and prolonged use of cyclophosphamide and corticosteroids. Although the efficacy of the therapeutic concept using cyclophosphamide and corticosteroids is well established, there have been some few reports that cyclophosphamide could be implicated in the genesis of malignancies. The pathophysiology of Wegener's granulomatosis should be better understood, and effective and less toxic alternative protocol should be established.     

Microbiologic depuration of Anadara tuberculosa (Mollusca: Arcidae)

In Costa Rica the mollusk Anadara tuberculosa represents a risk for human health due to the contamination of the growing waters and the fact that its is consumed raw. The families depending on the income obtained through commercialization of these animals have a low education and economic status. Therefore, it is of great importance to develop and evaluate simple methods of depuration that could be easily used by these families to make these mollusks safe for consumption. Bottles containing 11 of saline solution (25g/l) were prepared in duplicates to test the bactericidal effect of acetic acid. The solution in each bottle was adjusted to ph 4.5, 5.0 or 5.5 or held at ph of 7.0 or 8.0 for the controls. The solution in each bottle was then inoculated with approximately 1 X 104 cfu/ml of coliforms. Counts of coliforms were determined for each bottle 0, 1, 2, 4 and 8 hours after inoculation. For the depuration studies, specimens with diameters ranging from 4.0 to 4.5 cm were collected from a harvester at the estuary of Puntarenas, Gulf of Nicoya. Fifty specimens each were depurated in separate tanks containing 25 1 of oxygenated saline solution adjusted with acetic acid to an initial ph of 4.5 (treatment) or non adjusted ph of 8.0 (control). Counts of Enterobacteriaceae were determined, in duplicates, every 12 hr for 48 hr. An additional fifty animals were depurated using the defined method and tested to determine if they met international standards of microbiological quality for aerobic plate count, Enterobacteriaceae count, Escherichia coli count and presence of Salmonella. A sensory evaluation using a triangle test was performed to compare a typical dish prepared with depurated or non-depurated animals. A significant coliform reduction was determined in a saline solution (25 g/l) at a pH range of 4.5 to 5.5. This reduction, during 8 hr, was higher in the acid treatments compared to the controls. During depuration, the elimination of Enterobacteriaceae bacteria was faster when acetic was used (initial ph = 4.5) than when it was not. This elimination was more important the first 24 hr, time defined as adequate for the application of the method. The method has the advantage of transforming the bivalve in a product that is safe for human consumption, since it guarantees that the international standards of microbiological quality, for raw and depurated mollusks, are reached. On the other hand, the sensory qualities of a typical dish prepared with depurated animals are not affected by the method, which can be easily implemented and applied by the people that work in the extraction of this mollusk.     

Biochemical genetic studies on wild populations of the Vicia amoena complex (tribe Vicieae: Fabaceae)

A 45-year-old human immunodeficiency virus (HIV)-infected patient has suffered for a period of 4 years from recurrent and, later on, persistent oral pseudomembranous candidosis. The Candida isolates proved to be resistant to azole derivates in vitro and in vivo. Treatment with amphotericin B parenterally was successful in February 1996, but had to be stopped when chemotherapy for lymphoma was started. In August 1996, the patient showed a shift from the pseudomembranous to the erythematous type of oral candidosis; antiretroviral combination therapy including the HIV protease inhibitor saquinavir had been started 4 months previously. In July 1997, the patient was still suffering from a persistent oral candidosis of the erythematous type.     

Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease

Serum lipoprotein(a) (Lp(a)) levels were measured in 89 men with peripheral vascular disease (PVD) and 129 (100 male and 29 woman) healthy controls. Apolipoprotein(a) genetic polymorphism was determined by immunoblotting in all subjects. Patients with PVD had significantly higher serum Lp(a) levels than controls. Apolipoprotein(a) phenotype frequencies in patients with PVD did not differ from those of the control group. Both patients and controls with phenotype S2 had higher serum Lp(a) levels than those with phenotype S4. It should be emphasized that serum Lp(a) levels were significantly higher in PVD patients than controls for those with phenotype S2, S3/S4 and S4. Raised serum Lp(a) levels together with other lipoprotein abnormalities in patients with PVD imply a high cardiovascular risk. Genetic polymorphism clearly influences serum Lp(a) levels both in patients and controls. In patients with PVD, environmental and/or other genetic factors must play a role in raising Lp(a) levels.     

Characterisation of Trypanosoma cruzi populations by DNA polymorphism of the cruzipain gene detected by single-stranded DNA conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas' disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda)

The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions "h" and "p". Fraction "h" contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction "p" is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo.     

Molecular evidence for genetic subdivision of Antarctic krill (Euphausia superba Dana) populations

Antarctic krill (Euphausia superba Dana) is a key species in the Antarctic food web and occurs on a circumcontinental scale. Population genetic structure of this species was investigated by sequence analysis of the ND1 mitochondrial gene in four population samples collected at different geographical localities around the Antarctic continent. Results indicate the existence of significant genetic differences between samples, and we suggest that oceanographic barriers could be sufficiently strong and temporally stable to restrict gene flow between distinct areas. Moreover, our data indicate that Antarctic krill is not at mutation-drift equilibrium and that the species possibly has a low effective population size as compared to the census size.     

Microsatellite analysis of demographic genetic structure in fragmented populations of the tropical tree Symphonia globulifera

We developed genetic markers for three microsatellite loci in the tropical tree Symphonia globulifera and used them to examine the demographic genetic consequences of forest fragmentation. High levels of genetic variation were revealed in samples of adults, saplings, and seedlings. The more-variable loci exhibited less stability in allelic composition across sites and stages. The number of alleles per hectare (ha) of forest was similar when continuous forest plots were compared to plots from fragmented forest for all three stages. This pattern also held for the number of unique multilocus adult and sapling genotypes, but the number of unique seedling genotypes per ha of fragmented forest greatly exceeded expectations based on continuous forest data, probably due to the concentration of seeds into remnant forest patches by foraging bats. Significant inbreeding and genetic differentiation were most often associated with the fragmented forest and the seedlings. Finally, principal component analysis reaffirmed that a bottleneck, acting in concert with pre-existing genetic structure in the adults, had led to enhanced and rapid divergence in the seedlings following deforestation, a result that is of central interest for landscape management.     

Direct innervation of the mantle edge gland by neurosecretory axons in Helisoma duryi (Mollusca: Pulmonata)

The mantle edge gland of Helisoma duryi is innervated by neurosecretory axons from the pallial nerves. Synaptoid contacts occur between axons and gland cells, and there is ultrastructural evidence for the release of neurosecretory material. The mantle edge gland contributes to the deposition of periostracum during shell formation, and direct neurosecretory innervation may control shell growth and regeneration.     

The genus Myxidium (Protozoa: Myxosporida) in macrourid fishes

A physical model approach was used to investigate cholesterol gallstone dissolution kinetics in simulated bile. Critical experimental and theoretical investigations simulating in vivo conditions showed that, in the bile acid-lecithin solutions, there is a significant interfacial barrier for both cholesterol gallstone and cholesterol monohydrate pellet dissolution. In the present study, the rotating-disk dissolution method and the accompanying Levich theory were applied to assess the contributions of the diffusion convection mass transfer resistance and of the interfacial barrier to the overall kinetics. Cholesterol dissolution rates in bile acid solutions were about 2-20 times slower than diffusion-controlled rates depending upon the degree of agitation. As found in previous studies, these rates in the presence of sufficient concentrations of dissolution accelerators approached the theoretical diffusion-convection-controlled rates. To account for the much slower dissolution rates in bile acid-lecithin solutions, two possible kinetic interpretations were investigated. The first is based upon slow crystal-micellar solution interfacial kinetics, and the second is based upon a slow rate of cholesterol solubilization in the aqueous diffusion layer. For the latter, an analytical mathematical solution was obtained.     

Fine structure of the spermatozoa of Crassostrea gigas (Mollusca, Bivalvia)

STUDY OBJECTIVES: To compare the effectiveness of intramuscular meperidine (2 mg/kg) and promethazine (1 mg/kg) with chlorpromazine (MPC) or without chlorpromazine (MP) (1 mg/kg) for sedation of children undergoing emergency department procedures. DESIGN: Randomized, double-blind trial. SETTING: A community and university hospital ED. TYPE OF PARTICIPANTS: Eighty-seven hemodynamically and neurologically stable children less than 16 years old. INTERVENTIONS: IM sedation followed by intended procedure. MEASUREMENTS AND MAIN RESULTS: Children receiving either combination were not significantly different with regard to age, sex, weight, chronic illness, and indications. Procedures included laceration repair (46), fracture reduction (25), and others (16). Mean onset of action was similar (16 +/- 12 minutes), whereas the duration of action was significantly longer after MPC (63 +/- 57 minutes [mean +/- SD] compared with MP 29 +/- 36 minutes; P < .05, Student's t-test). Paradoxical hyperactivity occurred only after MP (three of 43 cases; P = NS, Fisher's exact test), whereas transient oxygen desaturation occurred only after MPC (one of 44 cases; P = NS). No other serious complications were observed. Three observers rated the effectiveness of sedation and analgesia on separate 10.2-cm visual-analog scales. Overall, MPC received significantly better ratings (7.4 +/- 2.1 cm) than MP (5.7 +/- 3.0 cm; P < .05, Mann-Whitney U test). Parents believed sedation worked well in 90% of cases. Their children had bad memories of the procedure in only 9% of cases. CONCLUSION: Elimination of chlorpromazine from the IM combination of meperidine and promethazine for pediatric sedation during ED procedures results in a significant reduction in efficacy.     

Cell lineage of the prototroch of Patella vulgata (Gastropoda, Mollusca)

The trochophore larva of the archaeogastropod mollusc Patella vulgata has a well-developed locomotory organ, the ciliated prototroch. This structure is formed from specific founder cells, the trochoblasts. Two methods were employed to determine the composition and cell lineage of the prototroch. Fluorescent cell-lineage tracer injection in trochoblasts and trochoblast founder cells was used to show how the various trochoblasts became incorporated into the prototroch. Scanning electron microscopy was used to study both differentiation, more specifically ciliation, of trochoblasts and localization of trochoblasts in the prototroch. The results obtained with both methods are in accordance with each other. During early development all trochoblasts involved in prototroch formation become cell cycle-arrested and develop cilia. Subsequently, the trochoblasts shift in position to form a circular prototroch and a number of trochoblasts deciliate. As a result of these processes the mature prototroch consists of a number of heavily ciliated cells as well as a number of deciliated cells. Comparison of prototrochs from a number of spiralian species shows that this structure is very conserved during evolution. The significance of this is discussed.     

The genetic relationship between the Finns and the Finnish Saami (Lapps): analysis of nuclear DNA and mtDNA

The genetic relationships between two Finno-Ugric-speaking populations, the Finns and the Finnish Saami (Lapps), were studied by using PCR for six nuclear-DNA marker loci, mitochondrial restriction-site polymorphism, and sequence variation of a 360-bp segment of the mitochondrial control region. The allele frequencies of each of the nuclear-DNA marker loci and the frequencies of mtDNA restriction haplotypes were significantly different between the populations. The Saami showed exceptionally low variation in their mtDNA restriction sites. The 9-bp deletion common in East Asian populations was not observed, nor did the haplotype data fit into the haplogroup categorization of Torroni et al. The average number of nucleotide substitutions from the mtDNA haplotype data indicated that the Finnish Saami may be closer to the Finns than to the other reference populations, whereas nuclear DNA suggested that the Finns are more closely related to the European reference populations than to the Finnish Saami. The similarity of the Finns to the other Europeans was even more pronounced according to the sequence data. We were unable to distinguish between the Finns and either the Swiss or Sardinian reference populations, whereas the Finnish Saami clearly stood apart. The Finnish Saami are distinct from other Circumarctic populations, although two of the lineages found among the Saami showed closer relationship to the Circumarctic than to the European lineages. The sequence data indicated an exceptionally high divergence for the Saami mtDNA control lineages. The distribution of the pairwise nucleotide differences in the Saami suggested that this population has not experienced an expansion similar to what was indicated for the Finns and the reference populations.     

Structural analysis of populations : new statistical approach to the problem of age in Squilla mantis (Crustacea Stomatopoda)

Thirty-three male length-frequency samples are analysed first by a multivariate procedure. The evolutionary cycles are pointed out by the interpretation of the first two components. The simulation model is built up on the basis of factorial analysis. The assessment of age classes parameters (mean, standard deviation, percentage) is then carried out by the simulation of data. Their configuration with respect to the two components points out the proximity of the observed and generated samples and shows the value of the statistical model.     

Hierarchical analysis of genetic structure in native fire ant populations: results from three classes of molecular markers

We describe genetic structure at various scales in native populations of the fire ant Solenopsis invicta using two classes of nuclear markers, allozymes and microsatellites, and markers of the mitochondrial genome. Strong structure was found at the nest level in both the monogyne (single queen) and polygyne (multiple queen) social forms using allozymes. Weak but significant microgeographic structure was detected above the nest level in polygyne populations but not in monogyne populations using both classes of nuclear markers. Pronounced mitochondrial DNA (mtDNA) differentiation was evident also at this level in the polygyne form only. These microgeographic patterns are expected because polygyny in ants is associated with restricted local gene flow due mainly to limited vagility of queens. Weak but significant nuclear differentiation was detected between sympatric social forms, and strong mtDNA differentiation also was found at this level. Thus, queens of each form seem unable to establish themselves in nests of the alternate type, and some degree of assortative mating by form may exist as well. Strong differentiation was found between the two study regions using all three sets of markers. Phylogeographic analyses of the mtDNA suggest that recent limitations on gene flow rather than longstanding barriers to dispersal are responsible for this large-scale structure.     

PCR-restriction fragment length polymorphism (RFLP) analyses reveal both extensive clonality and local genetic differences in Candida albicans

Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to obtain genotypes for the diploid pathogenic yeast, Candida albicans, we analysed 204 C. albicans isolates from three populations of the Duke University community: two from clinical sources [one from patients infected with human immunodeficiency virus (HIV) and the other from patients without HIV infection], and the third from healthy student volunteers. The results indicated: (i) extensive evidence for clonality within and between populations of C. albicans; and (ii) greater genotypic and gene diversities in the nonclinical population than those derived from clinical specimens, regardless of HIV status. The two clinical populations were genetically more similar to each other than either was to the population consisting of isolates from healthy people. Within each population sample there was a general lack of heterozygotes, and random associations of alleles within and between loci were found in less than 50% of the loci or pairs of loci. These findings were consistent between the two sets of samples analysed: those including all isolates and those including only clone-corrected isolates. Possible mechanisms are presented to explain the observed patterns of genetic variation within and between C. albicans populations.     

Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine

Nucleic acid immunization has been investigated as immunotherapy for infectious diseases as well as for treating specific types of cancers. In this approach, nucleic acid expression cassettes are directly inoculated into the host, whose transfected cells become the production source of novel and possibly immunologically foreign protein. We have developed a DNA vaccine construct which encodes for PSA by cloning a cDNA for PSA into a mammalian expression vector under control of a CMV promoter. We investigated and characterized the immunogenicity of PSA DNA expression cassettes in mice. PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation cytotoxic T lymphocyte (CTL), and flow cytometry assays. We observed a strong and persistent antibody response against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Using synthetic peptides spanning the PSA open frame, we identified four dominant T helper epitopes of PSA. Furthermore, immunization with PSA plasmid induced MHC Class I CD8+ T cell-restricted cytotoxic T lymphocyte response against tumor cell targets expressing PSA. The prostate represents a very specific functional organ critical for reproduction but not for the health and survival of the individual. Understanding the immunogenicity of PSA DNA immunization cassettes offers insight into the possible use of this tumor-associated antigen as a target for immunotherapy. These results demonstrate the ability of the genetic PSA to serve as a specific immune target capable of generating both humoral and cellular immune responses in vivo.     

Molecules and morphology: conflicts and congruence within the fern genus Trichomanes (Hymenophyllaceae)

The interaction between three independent data sets (anatomy/morphology, cytology, molecules) has been evaluated within the controversial genus Trichomanes (Hymenophyllaceae). Anatomy/morphology, cytology, and rbcL sequences, despite their high and significant level of incongruence, were thus empirically combined with differential weighting in a cladistic analysis within Trichomanes in order to give an appreciation of the contribution of each data set in the resulting topologies and to study more precisely the nature of potential conflicts. Results show that any standard statistics values (such as bootstrap) do not appear to be objectively useful for the choice of the "best" topology or the "good" clades provided by the combination. This weighting approach reveals three cases: (i) some clades (such as subgenus Didymoglossum) are always retrieved and correspond to the absence of conflicts between the different data, (ii) some new clades (such as subgenus Achomanes) are either provided or reenforced as a "synergetic" result of the combination of the data and (iii) that remaining conflicting clades reflect the persistence of incongruence between data whatever the weighting.