The preparation of holey films for electron microscopy

Zusammenfassung Vier verschiedene Methoden zur Gewinnung von Lochfolien für elektronenmikroskopische Belange werden beschrieben. Damit lassen sich unter anderem leicht Anzahl der Löcher pro Flächeneinheit und Lochdurchmesser variieren, so dass eine weitgehende Anpassung der Filmqualität an die sonstigen Versuchsbedingungen ermöglicht wird.     

Variability of El Niño/Southern Oscillation activity at millennial timescales during the Holocene epoch

The variability of El Ni?o/Southern Oscillation (ENSO) during the Holocene epoch, in particular on millennial timescales, is poorly understood. Palaeoclimate studies have documented ENSO variability for selected intervals in the Holocene, but most records are either too short or insufficiently resolved to investigate variability on millennial scales. Here we present a record of sedimentation in Laguna Pallcacocha, southern Ecuador, which is strongly influenced by ENSO variability, and covers the past 12,000 years continuously. We find that changes on a timescale of 2-8 years, which we attribute to warm ENSO events, become more frequent over the Holocene until about 1,200 years ago, and then decline towards the present. Periods of relatively high and low ENSO activity, alternating at a timescale of about 2,000 years, are superimposed on this long-term trend. We attribute the long-term trend to orbitally induced changes in insolation, and suggest internal ENSO dynamics as a possible cause of the millennial variability. However, the millennial oscillation will need to be confirmed in other ENSO proxy records.     

Sperm competition: motility and the midpiece in primates

In animals with multiple-partner mating systems, the gametes of two or more males must compete to fertilize a given set of ova. Here we show that the volume of the midpiece in individual sperm is significantly greater in primate species in which the females mate with multiple partners, and in which males have larger testes in relation to their body weight, than in those species that mate with only one partner and have relatively small testes. Our results indicate that sexual selection by sperm competition has influenced the evolution of a specific component of male-gamete morphology, the volume of the sperm midpiece.     

The bacteriophage straight phi29 portal motor can package DNA against a large internal force

As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells. Bacteriophage straight phi29 packages its 6.6-microm long, double-stranded DNA into a 42 x 54 nm capsid by means of a portal complex that hydrolyses ATP. This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor. This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5 microm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to approximately 50 pN owing to DNA confinement. Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection.     

Suppressing unwanted memories by executive control

Freud proposed that unwanted memories can be forgotten by pushing them into the unconscious, a process called repression. The existence of repression has remained controversial for more than a century, in part because of its strong coupling with trauma, and the ethical and practical difficulties of studying such processes in controlled experiments. However, behavioural and neurobiological research on memory and attention shows that people have executive control processes directed at minimizing perceptual distraction, overcoming interference during short and long-term memory tasks and stopping strong habitual responses to stimuli. Here we show that these mechanisms can be recruited to prevent unwanted declarative memories from entering awareness, and that this cognitive act has enduring consequences for the rejected memories. When people encounter cues that remind them of an unwanted memory and they consistently try to prevent awareness of it, the later recall of the rejected memory becomes more difficult. The forgetting increases with the number of times the memory is avoided, resists incentives for accurate recall and is caused by processes that suppress the memory itself. These results show that executive control processes not uniquely tied to trauma may provide a viable model for repression.     

Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis

Juvenile polyposis (JP; OMIM 174900) is an autosomal dominant gastrointestinal hamartomatous polyposis syndrome in which patients are at risk for developing gastrointestinal cancers. Previous studies have demonstrated a locus for JP mapping to 18q21.1 (ref. 3) and germline mutations in the homolog of the gene for mothers against decapentaplegic, Drosophila, (MADH4, also known as SMAD4) in several JP families. However, mutations in MADH4 are only present in a subset of JP cases, and although mutations in the gene for phosphatase and tensin homolog (PTEN) have been described in a few families, undefined genetic heterogeneity remains. Using a genome-wide screen in four JP kindreds without germline mutations in MADH4 or PTEN, we identified linkage with markers from chromosome 10q22-23 (maximum lod score of 4.74, straight theta=0.00). We found no recombinants using markers developed from the vicinity of the gene for bone morphogenetic protein receptor 1A (BMPR1A), a serine-threonine kinase type I receptor involved in bone morphogenetic protein (BMP) signaling. Genomic sequencing of BMPR1A in each of these JP kindreds disclosed germline nonsense mutations in all affected kindred members but not in normal control individuals. These findings indicate involvement of an additional gene in the transforming growth factor-beta (TGF-beta) superfamily in the genesis of JP, and document an unanticipated function for BMP in colonic epithelial growth control.     

Dominant effector genetics in mammalian cells

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.