The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.     

Isolation of the alpha 3-chain of human type V collagen and characterization by partial sequencing.

The alpha 1 alpha 2 alpha 3-chain form of human type V collagen was solubilized from placenta by pepsin treatment and isolated by ion-exchange chromatography. The alpha 3-chain was further separated after denaturation of the triple helix also by ion-exchange chromatography, cleaved with lysyl endopeptidase and the fragments separated by size-exclusion chromatography and reversed phase HPLC. N-Terminal sequence analysis of the fragments and comparison to sequences contained in a database indicated a relatively high similarity of the alpha 3(V)-chain to alpha 1(V) and alpha 1(XI) with an identity of approximately 73%.     

Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells.

The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

Calcaneus secundarius: description and frequency in six skeletal samples

The calcaneus secundarius (CS) is a small accessory ossicle of the anterior calcaneal facet. A sample of 1,367 calcanei including early 20th century U.S. blacks and whites, XIIth Dynasty Egyptians, Islamic Period Bahrainis, and protohistoric American Indians from two states were examined to determine the frequency of this nonmetric trait. Statistical testing of the correlation of the trait with sex could only be performed on the blacks and whites, a documented cadaver collection. Tests of chi 2 indicated that, although sex differences were insignificant, significant group (not necessarily racial or geographical) differences existed at the .05 level. There was no correlation of side dominance in the overall sample, although significant population differences existed. The trait frequencies of the black and white samples were more similar to one another than to the nonindustrial groups who, in turn, were more similar to each other than to the Terry sample. The findings suggest that a CS is of genetic or congenital origin resulting in a secondary center of ossification in the anterior calcaneal facet.     

New mechanical aid to physiotherapy in cystic fibrosis.

The force of impact and frequency of percussion by physiotherapists and parents of children with cystic fibrosis were analysed on a special test rig and incorporated in a prototype percussor. In adult volunteers and cadavers a maximum intrathoracic pressure could be achieved by a critical frequency of mechanical percussion which was higher than that reached by physiotherapists and parents. Consequently the percussor was redesigned to operate at this optimum frequency. It was then discovered that if the percussor was pressed firmly enough against the chest, this maximum intrathoracic pressure could be indicated by quivering of the voice. In a continuing study of intrathoracic pressures obtained mechanically and manually the Salford percussor''s produced higher pressures than the physiotherapists'' and maintained them constantly, while the physiotherapists'' efforts and results varied from one to another. Hospital and domiciliary use of the percussor have shown it to help in the first stage of the physiotherapy routine for patients with cystic fibrosis. The percussor should enable adolescents and adults to treat themselves and encourage twice-daily and more effective chest treatments. It is easy to apply and its speed and efficiency should enable parents to improve the quality of their chest therapy for younger children at home. Its long-term benefits are difficult to assess because of the nature of the disease.     

Moths complement bumblebee pollination of red clover: a case for day-and-night insect surveillance

Recent decades have seen a surge in awareness about insect pollinator declines. Social bees receive the most attention, but most flower-visiting species are lesser known, non-bee insects. Nocturnal flower visitors, e.g. moths, are especially difficult to observe and largely ignored in pollination studies. Clearly, achieving balanced monitoring of all pollinator taxa represents a major scientific challenge. Here, we use time-lapse cameras for season-wide, day-and-night pollinator surveillance of Trifolium pratense (L.; red clover) in an alpine grassland. We reveal the first evidence to suggest that moths, mainly Noctua pronuba (L.; large yellow underwing), pollinate this important wildflower and forage crop, providing 34% of visits (bumblebees: 61%). This is a remarkable finding; moths have received no recognition throughout a century of T. pratense pollinator research. We conclude that despite a non-negligible frequency and duration of nocturnal flower visits, nocturnal pollinators of T. pratense have been systematically overlooked. We further show how the relationship between visitation and seed set may only become clear after accounting for moth visits. As such, population trends in moths, as well as bees, could profoundly affect T. pratense seed yield. Ultimately, camera surveillance gives fair representation to non-bee pollinators and lays a foundation for automated monitoring of species interactions in future.     

Developability studies before initiation of process development

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2−8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.     

RNase P: role of distinct protein cofactors in tRNA substrate recognition and RNA-based catalysis

The Escherichia coli ribonuclease P (RNase P) has a protein component, termed C5, which acts as a cofactor for the catalytic M1 RNA subunit that processes the 5′ leader sequence of precursor tRNA. Rpp29, a conserved protein subunit of human RNase P, can substitute for C5 protein in reconstitution assays of M1 RNA activity. To better understand the role of the former protein, we compare the mode of action of Rpp29 to that of the C5 protein in activation of M1 RNA. Enzyme kinetic analyses reveal that complexes of M1 RNA–Rpp29 and M1 RNA–C5 exhibit comparable binding affinities to precursor tRNA but different catalytic efficiencies. High concentrations of substrate impede the activity of the former complex. Rpp29 itself exhibits high affinity in substrate binding, which seems to reduce the catalytic efficiency of the reconstituted ribonucleoprotein. Rpp29 has a conserved C-terminal domain with an Sm-like fold that mediates interaction with M1 RNA and precursor tRNA and can activate M1 RNA. The results suggest that distinct protein folds in two unrelated protein cofactors can facilitate transition from RNA- to ribonucleoprotein-based catalysis by RNase P.