SMN-mediated assembly of RNPs: a complex story

Although many RNA-protein complexes or ribonucleoproteins (RNPs) assemble spontaneously in vitro, little is known about how they form in the environment of a living cell. Insight into RNP assembly has come unexpectedly from functional analyses of the survival motor neuron (SMN) protein, a gene product that is affected in the neuromuscular disease spinal muscular atrophy. These studies show that the assembly of spliceosomal U-rich small nuclear RNPs in vivo depends on the activity of two large protein complexes, one of which contains the SMN protein. These complexes might also facilitate the assembly of other cellular RNPs.     

A technique for joint center analysis using a stored program calculator

For a long time in the study of joint kinematics, the instant center of rotation in plane motion was obtained through graphic drawings. Since then the study of joint kinematics has become three-dimensional involving the use of computers. For this paper a stored-program calculator has been used as it is a precise instrument and several films can be used even if their positions are very close to one another.

A movement is never perfectly plane, it was important to define a coefficient (in percentage) to qualify the more or less plane character of a movement.

We believe that an analytical location is a better way than using graphic drawings of I.C.R.:

1. (1) to smooth the raw coordinates;

2. (2) to calculate the plane motion coefficient in order to eliminate an X-Ray picture of a whole series of pictures for lack of plane character;

3. (3) to define in the results an error rectangle whose dimensions are linked to errors in the observation and then to pick out among the points of a body those with the smallest risk for error.

To probe this method the two radio-ulnaris joints have been studied. At present studies are being carried on to compare the I.C.R.'s behaviour of the lumbar spine during a motion of lateral inflexion both in the case of normal people and people with scoliosis.     

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

We compared the expression of a functional recombinant TMVspecific fullsize antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of fullsize rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The fullsize rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional fullsize rAb29 expression were high in the apoplast (up to 8.5g per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as fullsize rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMVcoat protein monomers as rAb29.     

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.     

A novel Fomitiporia species associated with esca on grapevine in South Africa

Esca disease of grapevine is a complex trunk disease consisting of several symptoms, one of which, white rot, has been found to be caused by various basidiomycetes within the order Hymenochaetales. During recent surveys of esca-related pathogens in South African vineyards, several unidentified basidiomycetes were isolated from white rot occurring in diseased vines. A new Fomitiporia species, F. capensis, is described based on fruit body morphology and combined internal transcribed spacer (ITS) and large-subunit (LSU) phylogeny, where it forms a clearly delineated and well-supported clade. Morphologically, F. capensis is similar to F. punctata in that both species essentially lack setae. Fomitiporia capensis, F. punctata and F. aethiopica produce similarly sized basidiospores but differ in terms of host range, pore size and, possibly, fruit body shape. Phylogenetically, F. capensis appears to be related to F. tenuis, though morphologically, the species differ significantly in that F. tenuis has smaller pores and smaller basidiospores. Fomitiporia capensis was found to occur widely as vegetative mycelium throughout the Western Cape Province, though fruit bodies were scarce in comparison. A vineyard with fruit bodies was also found in Limpopo in the north east of the country. Fruit bodies were found growing on the underside of the cordon of living vines displaying external symptoms typically associated with esca, or general decline and dieback symptoms together with internal white rot.     

High-throughput phenotypic assessment of cardiac physiology in four commonly used inbred mouse strains

Mice with genetic alterations are used in heart research as model systems of human diseases. In the last decade there was a marked increase in the recognition of genetic diversity within inbred mouse strains. Increasing numbers of inbred mouse strains and substrains and analytical variation of cardiac phenotyping methods require reproducible, high-throughput methods to standardize murine cardiovascular physiology. We describe methods for non-invasive, reliable, easy and fast to perform echocardiography and electrocardiography on awake mice. This method can be used for primary screening of the murine cardiovascular system in large-scale analysis. We provide insights into the physiological divergence of C57BL/6N, C57BL/6J, C3HeB/FeJ and 129P2/OlaHsd mouse hearts and define the expected normal values. Our report highlights that compared to the other three strains tested C57BL/6N hearts reveal features of heart failure such as hypertrophy and reduced contractile function. We found several features of the mouse ECG to be under genetic control and obtained several strain-specific differences in cardiac structure and function.     

Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells

Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin™ is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin™ could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin™ were generated. Immunoblot experiments showed that Solulin™ accumulated to maximum levels of 115 and 27 μg g⁻¹ plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin™ was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin™ from the BY2 culture supernatant. The sequence was identical to that of Solulin™ produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.     

Ordination of breeding birds in relation to environmental gradients in three southeastern United States floodplain forests

We used an ordination approach to identify factors important to the organization of breeding bird communities in three floodplains: Cache River, Arkansas (AR), Iatt Creek, Louisiana (LA), and the Coosawhatchie River, South Carolina (SC), USA. We used 5-min point counts to sample birds in each study area each spring from 1995 to 1998, and measured ground-surface elevations and a suite of other habitat variables to investigate bird distributions and community characteristics in relation to important environmental gradients. In both AR and SC, the average number of Neotropical migrant species detected was lowest in semipermanently flooded Nyssa aquatica Linnaeus habitats and greatest in the highest elevation floodplain zone. Melanerpes carolinus Linnaeus, Protonotaria citrea Boddaert, Quiscalus quiscula Linnaeus, and other species were more abundant in N. aquatica habitats, whereas Wilsonia citrina Boddaert, Oporornis formosus Wilson, Vireo griseus Boddaert, and others were more abundant in drier floodplain zones. In LA, there were no significant differences in community metrics or bird species abundances among forest types. Canonical correspondence analyses revealed that structural development of understory vegetation was the most important factor affecting bird distributions in all three study areas; however, potential causes of these structural gradients differed. In AR and SC, differences in habitat structure were related to the hydrologic gradient, as indexed by ground-surface elevation. In LA, structural variations were related mainly to the frequency of canopy gaps. Thus, bird communities in all three areas appeared to be organized primarily in response to repeated localized disturbance. Our results suggest that regular disturbance due to flooding plays an important role in structuring breeding bird communities in floodplains subject to prolonged inundation, whereas other agents of disturbance (e.g., canopy gaps) may be more important in headwater systems subject to only short-duration flooding. Management for avian community integrity in these systems should strive to maintain forest zonation and natural disturbance regimes.