A trnL-F based phylogeny for species ofPelargonium (Geraniaceae) with small chromosomes

Phylogenetic analysis was performed of 921 positions of trnL (UAA) 5 exon — trnF (GAA) exon chloroplast DNA regions from 68 representatives ofPelargonium sectt.Campylia, Cortusina, Glaucophyllum, Hoarea, Isopetalum, Ligularia, Otidia, Pelargonium, Peristera, Polyactium, andReniformia, together with five putative outgroup species from sectionsCiconium, Chorisma andJenkinsonia. The total data set therefore comprised 67.2 kb of DNA sequence. Two main ingroup clades were identified: one clade contains sectionsPeristera, Reniformia, andIsopetalum, the other contains sectionsCampylia, Cortusina, Glaucophyllum, Hoarea, Ligularia, Otidia, Pelargonium, Polyactium and two species currently grouped in sect.Peristera. Branching order among five main clades within the latter clade was not resolved. The trnL-F sequence data support monophyly only for sectionsReniformia andHoarea, the remainder of the currently recognized sections ofPelargonium being either paraphyletic or polyphyletic. The data further suggest that sect.Polyactium is diphyletic and that sect.Glaucophyllum is nested within sect.Pelargonium. One relatively derived clade, which represents half of the genus, contains predominantly geophytic and succulent species, occurring in the geographically restricted winter rainfall region of the South African Cape. This pattern is interpreted as reflecting explosive radiation, possibly as an adaptive response to recent aridification in the western Cape.     

Nuclear mitochondrial pseudogenes as molecular outgroups for phylogenetically isolated taxa: a case study in Sphenodon

'Living fossil' taxa, by definition, have no close relatives, and therefore no outgroup to provide a root to phylogenetic trees. We identify and use a molecular outgroup in the sole extant lineage of sphenodontid reptiles, which separated from other reptiles 230 million years ago. We isolated and sequenced a partial nuclear copy of the mitochondrial cytochrome b gene. We confirm the copy is indeed not mitochondrial, is older than all extant mitochondrial copies in Sphenodon (tuatara), and is therefore useful as a molecular outgroup. Under phylogenetic analysis, the nuclear copy places the root of the tuatara mitochondrial gene tree between the northern and the southern (Cook Strait) groups of islands of New Zealand that are the last refugia for Sphenodon. This analysis supports a previous mid-point rooted mitochondrial gene tree. The mitochondrial DNA tree conflicts with allozyme analyses which place a Cook Strait population equidistant to all northern and other Cook Strait populations. This population on North Brother Island is the only natural population of extant S. guntheri; thus, we suggest that the current species designations of tuatara require further investigation.     

The nucleotide transporter of Caedibacter caryophilus exhibits an extended substrate spectrum compared to the analogous ATP/ADP translocase of Rickettsia prowazekii

The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol. These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared. The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter. ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter. Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors. Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of [alpha-32P]ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter).     

Thalidomide inhibits early atherogenesis in apoE-deficient mice

Inflammation is present in all stages of atherosclerosis, from fatty streaks to rupture of mature plaques. Tumour necrosis factor (TNF)-alpha is expressed in atherosclerotic lesions but its role in atherogenesis has not been defined. To clarify the role of this cytokine, we administered thalidomide, a compound known to inhibit TNF-alpha production, to homozygous apolipoprotein E-deficient (apoE-/-) mice in order to examine the effect of thalidomide on the development of early atherosclerotic lesions. Twelve apoE-/- mice were randomized to receive either sustained-release thalidomide or placebo pellets implanted subcutaneously, and the amount of atherosclerosis was quantified six weeks later. Thalidomide was well tolerated and did not result in any changes in body weight. Mice treated with thalidomide had significantly smaller mean (7986 +/- 5189 vs 19607 +/- 10353 microns 2, p = 0.05) and maximum (15800 [12777-23675] vs 37169 [28000-41351] microns 2, p = 0.03) lesion sizes than those treated with placebo. Thus, thalidomide is capable of inhibiting the early development of atherosclerosis, presumably by inhibition of TNF-alpha secretion.     

Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics

The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.     

Macrophage-specific expression of class A scavenger receptors enhances granuloma formation in the absence of increased lipid deposition.

Class A scavenger receptors (SR-A) have several proposed functions that could impact atherosclerosis and inflammatory processes. To define the function of SR-A in vivo, we created C57BL/6 transgenic mice that expressed bovine SR-A under the control of the restricted macrophage promoter, lysozyme (lyso-bSR-A). bSR-A mRNA was present in cultured peritoneal macrophages of transgenic mice and tissues that contain significant macrophages including spleen, lung, and ileum. Functional overexpression of SR-A was demonstrated in peritoneal macrophages both by augmented cholesterol ester deposition in response to AcLDL and enhanced adhesion in transgenic mice compared with nontransgenic littermates. To determine whether macrophage-specific expression of bSR-A regulated inflammatory responses, granulomas were generated by subcutaneous injection of carrageenan. Granuloma size was significantly increased in lyso-bSR-A transgenic mice compared with wild-type littermates [421 +/- 51 mg (n = 11) vs. 127 +/- 22 mg (n = 10), P < 0.001]. However, the larger granulomas in lyso-bSR-A transgenic mice were only associated with an increase in unesterified cholesterol, and not cholesterol esters. Furthermore, granulomas from transgenic mice had an increase in the number of macrophages within the tissue.Therefore, macrophage expression of bSR-A increased presence of this cell type in granulomas without enhancing the deposition of cholesterol esters, consistent with a role of the adhesive property of the protein.     

Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence from a Phylogenetic Analysis of Internal Transcribed Spacer Sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon.Saprolegniaemerges as the most basal genus, sister toAchlya, Thraustotheca,andDictyuchus. AchlyaandThraustothecaare most closely related, whileDictyuchusappears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited bySaprolegniarepresents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall.     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma

athanassiadou p.p., athanassiades p. h., daffaris p., petrakakou e. i., fflerffa ch. i. and kffirkou k. a. (1998) Cytopathology 9, 240–247
Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma
The aim of this study was to add to existing information on the effects of certain tumour markers expressed by breast cancers on tumour malignancy as evidenced by size of primary and occurrence of lymph node invasion. One hundred freshly resected breast cancers were examined by immunocytochemical staining of imprint smears for Cathepsin D and pS2. Oestrogen receptor (ER) and progesterone receptor (PR) were tested for by dextrose-coated charcoal (DCC) assay and the results correlated with tumour size, histology and presence or absence of lymph node metastases at the time of surgery using χ² analysis. A significant positive correlation was demonstrated between Cathepsin D positivity and ER, PR and pS2 positivity. In tumours < 2 cm in diameter at surgery a positive correlation was observed between Cathepsin D positivity and the presence of lymph node metastases. The findings support the hypothesis that Cathepsin D may promote early metastasis, possibly by its proteolytic activity.