Novel dominant mutations in Saccharomyces cerevisiae MSH6

Inherited mutations in the mismatch repair (MMR) genes MSH2 and MLH1 are found in most hereditary nonpolyposis colon cancer (HNPCC) patients studied. Eukaryotic MMR uses two partially redundant mispair-recognition complexes, Msh2p-Msh6p and Msh2p-Msh3p (ref.2) Inactivation of MSH2 causes high rates of accumulation of both base-substitution and frameshift mutations. Mutations in MSH6 or MSH3 cause partial defects in MMR, with inactivation of MSH6 resulting in high rates of base-substitution mutations and low rates of frameshift mutations; inactivation of MSH3 results in low rates of frameshift mutations. These different mutator phenotypes provide an explanation for the observation that MSH2 mutations are common in HNPCC families, whereas mutations in MSH3 and MSH6 are rare. We have identified novel missense mutations in Saccharomyces cerevisiae MSH6 that appear to inactivate both Msh2p-Msh6p- and Msh2p-Msh3p-dependent MMR. Our work suggests that such mutations may underlie some cases of inherited cancer susceptibility similar to those caused by MSH2 mutations.     

Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.     

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.     

Reversal of pathological pain through specific spinal GABAA receptor subtypes

Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal gamma-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA(A) receptors should be able to compensate for this loss. With the use of GABA(A)-receptor point-mutated knock-in mice in which specific GABA(A) receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA(A) receptors containing the alpha2 and/or alpha3 subunits. We show that their selective activation by the non-sedative ('alpha1-sparing') benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.     

Do female hyaenas choose mates based on tenure?

In their investigation into whether female mate-choice drives male dispersal, H?ner et al. argue that female spotted hyaenas (Crocuta crocuta) prefer mates whose tenure in the social group is less than the females' age, to avoid paternal incest, and suggest that male dispersal reflects this preference. However, we are not persuaded that females choose mates on the basis of tenure because H?ner et al. overlook the alternative hypothesis that dispersal status itself is important in female mate-choice, such that females prefer immigrants over natal males. Like mate-choice based on tenure, choice based on dispersal status reduces the risk of incest.     

Relationship between the microstructure and properties of thermomechanically processed Fe-17Mn and Fe-17Mn-3Al steels

Two austenitic Mn steels(Fe-17 Mn and Fe-17 Mn-3 Al(wt%, so as the follows)) were subjected to thermomechanical processing(TMP) consisting of forging followed by solutionization and hot rolling. The rolled samples were annealed at 650 and 800°C to relieve the internal stress and to induce recrystallization. The application of TMP and heat treatment to the Fe-17 Mn/Fe-17 Mn-3 Al steels refined the austenite grain size from 169 μm in the as-solutionized state to 9–13 μm, resulting in a substantial increase in hardness from HV 213 to HV 410 for the Fe-17 Mn steel and from HV 210 to HV 387 for the Fe-17 Mn-3 Al steel. The elastic modulus values, as evaluated by the nanoindentation technique, increased from(175 ± 11) to(220 ± 12) GPa and from(163 ± 15) to(205 ± 13) GPa for the Fe-17 Mn and Fe-17 Mn-3 Al steels, respectively. The impact energy of the thermomechanically processed austenitic Mn steels was lower than that of the steels in their as-solutionized state. The addition of Al to the Fe-17 Mn steel decreased the hardness and elastic modulus but increased the impact energy.     

The first vertebrate fossil from Socotra Island (Yemen) is an early Holocene Egyptian fruit bat

Data on the prehistoric biota of the Socotra Archipelago (Yemen) are scarce. We report on the first extinct vertebrate ever recorded from this western Indian Ocean island group. An exceptionally preserved adult specimen of Egyptian fruit bat Rousettus aegyptiacus was found in a calcite flowstone in Hoq Cave on the NE coast of Socotra Island. Accelerator mass spectrometry (AMS) radiocarbon dating on bone fragments estimates the fossil at ca. 7600–7400 cal a BP (radiocarbon age 6669 ± 70 ¹⁴C a BP) corresponding to an early Holocene wet period in southern Arabia and the Horn of Africa. Morphometric analysis of the skull compared to Recent specimens from the neighbouring mainlands finds the fossil morphologically most similar to Egyptian (Nile Valley and Libyan Desert), not to East African or Middle Eastern populations. The species is currently absent from the Horn of Africa (Somalia and Eastern Ethiopia). Although potentially a stray individual, we also discuss the palaeoenvironmental implications under the hypothetical scenario that the fruit bat is a remnant of an extinct resident population. If so, the extinction of an important pollinator and seed disperser may have impacted the Socotran terrestrial ecosystems.