Rescue of influenza C virus from recombinant DNA

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3' and 5' noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10(3) and 10(4) PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.     

Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements

Glypican (GPC)-3 inhibits cell proliferation and regulates cell survival during development. This action is demonstrated by GPC3 loss-of-function mutations in humans and mice. Here, we show that the GPC3 core protein is processed by a furinlike convertase. This processing is essential for GPC3 modulating Wnt signaling and cell survival in vitro and for supporting embryonic cell movements in zebrafish. The processed GPC3 core protein is necessary and sufficient for the cell-specific induction of apoptosis, but in vitro effects on canonical and noncanonical Wnt signaling additionally require substitution of the core protein with heparan sulfate. Wnt 5A physically associates only with processed GPC3, and only a form of GPC3 that can be processed by a convertase is able to rescue epiboly and convergence/extension movements in GPC3 morphant embryos. Our data imply that the Simpson-Golabi-Behmel syndrome may in part result from a loss of GPC3 controls on Wnt signaling, and suggest that this function requires the cooperation of both the protein and the heparan sulfate moieties of the proteoglycan.     

Characterization of immune responses during infection with Mycobacterium avium strains 100, 101 and the recently sequenced 104

Mycobacterium avium strain 104 was chosen as the M. avium isolate to sequence, as it is virulent to humans, stable and readily transfectable. As this strain has not been widely studied we sought to investigate the pattern of 104 infection in mice. Bacterial growth and the immune response generated were compared with infection with the low virulence M. avium strain 100, and the high virulence common laboratory strain, 101. Mycobacterium avium strains 104 and 101 grew progressively within mice, while strain 100 was gradually cleared. Strains 104 and 101 induced strong T cell activation and spleen cell cultures produced similar levels of IFN-gamma. In mice infected with strain 100 no significant T cell activation or IFN-gamma production was measured. Further, mice infected with strain 104 or 101 also displayed comparable inflammatory responses and similar granuloma formation, while only minimal inflammation was seen in mice infected with strain 100. Strains 101 and 104 also grew in a similar fashion in bone-marrow-derived macrophages and induced significant levels of TNF and nitric oxide. Thus infection with M. avium strain 104 induced an immunological response comparable to M. avium strain 101 and, with the availability of its sequence, should be a useful tool for designing new vaccines or drugs therapies to treat the increasing incidence of M. avium infection in humans.     

The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway

The Arabidopsis thaliana (L.) Heynh. mutant delayed-dehiscence2-2 (dde2-2) was identified in an En1/Spm1 transposon-induced mutant population screened for plants showing defects in fertility. The dde2-2 mutant allele is defective in the anther dehiscence process and filament elongation and thus exhibits a male-sterile phenotype. The dde2-2 phenotype can be rescued by application of methyl jasmonate, indicating that the mutant is affected in jasmonic acid biosynthesis. The combination of genetic mapping and a candidate-gene approach identified a frameshift mutation in the ALLENE OXIDE SYNTHASE (AOS) gene, encoding one of the key enzymes of jasmonic acid biosynthesis. Expression analysis and genetic complementation of the dde2-2 phenotype by overexpression of the AOS coding sequence confirmed that the male-sterile phenotype is indeed caused by the mutation in the AOS gene.     

Plasma levels of cortisol and oxytocin,and uterine activity after cervical artificial insemination in the ewe

The objective was to compare in the ewe the effects of easy and difficult procedures for artificial insemination (AI) (as related to rapid or poor accessibility of the cervix, respectively) on plasma cortisol (CORT) and oxytocin (OT), and uterine motility. All AI were simulated using a catheter empty of semen to study genital and environmental stimuli only. In experiment 1, 40 ewes were sampled after Al, and whether it was an easy or difficult procedure was reported for each animal. While CORT concentrations rose to a similar amount in all ewes, whatever the Al procedure, a significant OT response occurred after a difficult procedure only (n = 18) (17.4 +/- 1.7 versus 12.7 +/- 0.7 pg x mL(-1) before Al, p < 0.05). In experiment 2, uterine activity was monitored in 4 ewes using an implantable telemetric transmitter equipped with an intrauterine pressure catheter. An increased uterine activity occurred during 2 +/- 1 min after an easy Al (n = 5), whereas the evoked activity lasted for 15 +/- 4 min after a difficult Al (p < 0.001, n = 7). A similar long-lasting response occurred after OT administration (100 mIU, i.v.). We concluded that the increase in uterine motility after a difficult Al resulted from a reflex release of OT, and not to a "stress" effect.     

Science and society: genetic counselling and customary consanguineous marriage

Consanguineous marriage is customary in many societies, but leads to an increased birth prevalence of infants with severe recessive disorders. It is therefore often proposed that consanguineous marriage should be discouraged on medical grounds. However, several expert groups have pointed out that this proposal is inconsistent with the ethical principles of genetic counselling, overlooks the social importance of consanguineous marriage and is ineffective. Instead, they suggest that the custom increases the possibilities for effective genetic counselling, and recommend a concerted effort to identify families at increased risk, and to provide them with risk information and carrier testing when feasible.     

Lipid rafts, flotillin-1 and Alzheimer disease

A beta peptide accumulates in the extracellular space during Alzheimer's disease. It is the cleavage product of APP (Amyloid Precursor Protein), a large transmembrane protein. After ultracentrifugation, APP is found in a low-density fraction, enriched in cholesterol. These properties are characteristic of lipid rafts, which are microdomains that "float" like rafts on the plasma membrane. We have confirmed the presence of cholesterol in the core of the senile plaque, using the fluorescent probe filipin. In addition, we have shown that flotillin-1, a marker of rafts, accumulated in lysosomes of neurons in Alzheimer's disease. In most cases (76% of the flotillin-1 positive neurons), the accumulation was associated with the presence of neurofibrillary tangles. Our data suggest that the A beta peptide, which is poorly soluble in water, is actually linked with cholesterol, possibly from cellular membranes, in the extracellular space.