Testing why the sex of the maternal parent affects seedling survival in a gynodioecious species

In gynodioecious plants, seed offspring from hermaphrodites often perform less well than those from females. This lower performance sometimes can be attributed to inbreeding by hermaphrodites or to relatively greater provisioning of individual seeds by females. However, these hypotheses are not explanatory when only outcrossing occurs and when individual seeds of the two morphs are equally well provisioned. Three hypotheses may explain the lower fitness of seed offspring from hermaphrodites in such cases. The morphology hypothesis states that the opportunity for gametophytic selection is lower within flowers of hermaphrodites compared to flowers on females, because the perfect flowers of hermaphrodites are relatively short-styled. The cytotype hypothesis states that the performance difference is directly caused by an individual's cytotype, whose frequency in the population may differ for the two sex morphs. The pleiotropy hypothesis states that negative pleiotropic effects of nuclear restorer alleles or alleles hitchhiking with them are expressed more often by offspring from hermaphrodites. We performed two experiments using the gynodioecious plant Silene acaulis to contrast these hypotheses. In our first experiment we contrasted the morphology and pleiotropy hypotheses by performing controlled pollinations and subsequently planting seeds in both the greenhouse and field. Hermaphrodites of S. acaulis can produce both pistillate and perfect flowers, which allowed us to determine whether flower morphology affects offspring survivorship independent of the sex of the maternal parent. We found that neither seed mass nor germination differed between seeds from females and hermaphrodites. Offspring from pistillate flowers on hermaphrodites did not differ significantly in their survival compared to offspring from perfect flowers on hermaphrodites, but had lower survivorship compared to offspring from pistillate flowers on females, refuting the morphology hypothesis. In a second experiment, we compared offspring survival of full-sibling pairs of females and hermaphrodites (who shared the same cytoplasm) to contrast the cytotype and pleiotropy hypotheses. We found that seed offspring from females and hermaphrodites that shared the same cytoplasm differed in their survival, which is counter to the prediction of the cytotype hypothesis. In both experiments, the sex of the maternal parent significantly affected offspring survival, with seed offspring from hermaphrodites surviving less well than those from females. These results support the pleiotropy hypothesis. We conclude by discussing alternative ways of thinking about negative pleiotropic effects of nuclear restorers or "the cost of restoration."     

Identification and mutational analysis of bacteriophage PRD1 holin protein P35

Holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. We describe the identification of the membrane-containing phage PRD1 holin gene (gene XXXV). The PRD1 holin protein (P35, 12.8 kDa) acts similarly to its functional counterpart from phage lambda (gene S), and the defect in PRD1 gene XXXV can be corrected by the presence of gene S of lambda. Several nonsense, missense, and insertion mutations in PRD1 gene XXXV were analyzed. These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis.     

Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.     

Adhesion of monocytes to medical steel as used for vascular stents is mediated by the integrin receptor Mac-1 (CD11b/CD18; alphaM beta2) and can be inhibited by semiconductor coating

Implantation of stents into stenosed arteries helps to restore normal blood flow in ischemic organs. However, limited biocompatibility of the applied medical steel can cause acute thrombosis and long-term restenosis. Adhesion of monocytes to stent metal may participate in those acute and long-term complications of stent placement. Based on described prominent electrochemical properties of the interaction between the monocyte integrin receptor Mac-1 and its various ligands, we hypothesized, that this receptor is a central mediator of monocyte adhesion to stent metal and that semiconductor coating of medical steel reduces monocyte adhesion. Adhesion of monocytes on L-316 stainless steel was directly evaluated by light microscopy. Mac-1 could be identified as mediator of monocyte adhesion, since cell adhesion could be blocked by anti-Mac-1-antibodies, including the cross-reacting anti-GPIIb/IIIa antibody fragment abciximab. To further prove the central role of Mac-1, two CHO cell lines were generated expressing recombinant Mac-1 either as wild type, resulting in a low affinity receptor, or mutant with a GFFKR deletion of the alpha(M) subunit, resulting in a high affinity receptor. Indeed, adhesion was specific for Mac-1 and dependent on the affinity state of this integrin. Finally, we could demonstrate that Mac-1-mediated adhesion of monocytes to stents can be significantly inhibited by silicon carbide coating of the stent metal. In conclusion, the integrin Mac-1 and its affinity state could be identified as major mediators of monocyte adhesion on medical steel. As therapeutic strategies, the blockade of Mac-1 by antibodies or silicon carbide coating of steel inhibits monocyte adhesion on stents.     

A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer

CHEK2 (previously known as "CHK2") is a cell-cycle-checkpoint kinase that phosphorylates p53 and BRCA1 in response to DNA damage. A protein-truncating mutation, 1100delC in exon 10, which abolishes the kinase function of CHEK2, has been found in families with Li-Fraumeni syndrome (LFS) and in those with a cancer phenotype that is suggestive of LFS, including breast cancer. In the present study, we found that the frequency of 1100delC was 2.0% among an unselected population-based cohort of 1,035 patients with breast cancer. This was slightly, but not significantly (P=.182), higher than the 1.4% frequency found among 1,885 population control subjects. However, a significantly elevated frequency was found among those 358 patients with a positive family history (11/358 [3.1%]; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.11-4.63; P=.021, compared with population controls). Furthermore, patients with bilateral breast cancer were sixfold more likely to be 1100delC carriers than were patients with unilateral cancer (95% CI 1.87-20.32; P=.007). Analysis of the 1100delC variant in an independent set of 507 patients with familial breast cancer with no BRCA1 and BRCA2 mutations confirmed a significantly elevated frequency of 1100delC (28/507 [5.5%]; OR 4.2; 95% CI 2.4-7.2; P=.0002), compared with controls, with a high frequency also seen in patients with only a single affected first-degree relative (18/291 [6.2%]). Finally, tissue microarray analysis indicated that breast tumors from patients with 1100delC mutations show reduced CHEK2 immunostaining. The results suggest that CHEK2 acts as a low-penetrance tumor-suppressor gene in breast cancer and that it makes a significant contribution to familial clustering of breast cancer-including families with only two affected relatives, which are more common than families that include larger numbers of affected women.     

The soft triangle revisited

Five cadaveric adult human noses were examined to further elucidate the region of the soft triangle of the nose. Three distinct zones were found. Zone 1, just caudad to the lower lateral cartilages, consists of the fibers of the dilator nares anterior as it inserts into the dermis. Zone 2 is the dermal layer. Zone 3 is at the nostril rim and consists of muscle fibers interdigitating within the dermis; laterally, these fibers are continuations of the nasalis muscle, whereas medially they are extensions of the depressor septii muscle. Perpendicular incisions through any of these zones may cause notching and tip deformity. Incisions in zone 1 may, in addition, alter external nasal valve function by interrupting the fibers of the dilator naris anterior.     

Bacteria associated with hazelnut (Corylus avellana L.) decline are of two groups: Pseudomonas avellanae and strains resembling P. syringae pv. syringae

A total of 118 fluorescent pseudomonads associated with hazelnut decline, which has been occurring for many years in different areas of northern Greece and Italy, were assessed by performing a repetitive PCR analysis with enterobacterial repetitive intergenic consensus, box element, and repetive extragenic palindromic primer sets, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein extracts, a carbon compound utilization analysis, and an analysis to determine the presence of the syrB gene. A subset of 53 strains was also characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA) by using nine restriction endonucleases. The virulence of 40 representative strains was assessed by using serial doses. The pathogenic specificities of the strains were also verified. ARDRA carried out with HinfI revealed two main groups of strains, groups A and B, which exhibited a level of similarity of 57%. The other eight restriction endonucleases used did not separate the strains. In addition, a cluster analysis performed by the unweighted pair group method using arithmetic averages after repetitive PCR and SDS-PAGE of protein extracts also revealed the same two groups. Furthermore, the differential utilization of some carbon compounds made it possible to differentiate the groups. Virulence assessment clearly indicated that the group A strains are very virulent, whereas the group B strains proved to be mildly virulent for hazelnut. Group A included the strains isolated in northern Greece and central Italy (i.e., the province of Viterbo); these strains do not have the syrB gene, are pathogenically restricted to Corylus avellana, and belong to Pseudomonas avellanae. Group B includes the other strains obtained from hazelnut cultivated in Piedmont, Campania, Latium, Sicily, and Sardinia. They represent a distinct taxon closely related to Pseudomonas syringae pv. syringae.     

Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line

Mutations in alpha-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant alpha-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. alpha-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant alpha-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human alpha-synuclein. Lentivirus-induced overexpression of A53T mutant alpha-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant alpha-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant alpha-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in alpha-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.