The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Hyperoxia causes increased albumin permeability of cultured endothelial monolayers

The effect of phasic eye movement activity on ventilation during rapid-eye-movement (REM) sleep was studied in seven healthy young adults by use of the respiratory inductive plethysmograph. Mean ventilation (VE) and ventilatory components during REM sleep were not significantly different from that seen in either stages 1-2 or 3-4 sleep. The percent of rib cage contribution to ventilation in REM sleep, 29.3 +/- 5.1%, was reduced compared with 54.4 +/- 5.8% in stage 1-2 and 52.2 +/- 4.3% in stage 3-4 sleep (P less than 0.005). When one separated breaths by the degree of associated phasic eye movement activity, it became apparent that breathing during REM sleep is very heterogeneous. Increasing eye movement activity was associated with inhibition of ventilation with a reduction in VE from 5.1 +/- 0.3 to 3.8 +/- 0.3 l/min. Tidal volume and frequency both fell, whereas inspiratory duration was unchanged. Compartmental ventilation was also affected, with the fall in the rib cage contribution from 37.8 +/- 6.4 to 15.3 +/- 5.6%. Chest wall and abdominal movement became more asynchronous as phasic-eye-movement activity increased and frank paradoxical breathing was seen.     

On the Structure-Bounded Growth Processes in Plant Populations

If growing cells in plants are considered to be composed of increments (ICs) an extended version of the law of mass action can be formulated. It evidences that growth of plants runs optimal if the reaction–entropy term (entropy times the absolute temperature) matches the contact energy of ICs. Since these energies are small, thermal molecular movements facilitate via relaxation the removal of structure disturbances. Stem diameter distributions exhibit extra fluctuations likely to be caused by permanent constraints. Since the signal–response system enables in principle perfect optimization only within finite-sized cell ensembles, plants comprising relatively large cell numbers form a network of size-limited subsystems. The maximal number of these constituents depends both on genetic and environmental factors. Accounting for logistical structure–dynamics interrelations, equations can be formulated to describe the bimodal growth curves of very different plants. The reproduction of the S-bended growth curves verifies that the relaxation modes with a broad structure-controlled distribution freeze successively until finally growth is fully blocked thus bringing about “continuous solidification”.     

Isolation and analysis of monoclonal antibodies against various gangliosides

Female BALB/c mice were immunized with human melanoma (Mewo) cells containing ganglioside GD3 as a surface antigen. Immune splenocytes were fused with syngeneic P3-X63.Ag 8 myeloma cells. Antibodies produced by hybrid clones were analyzed by solid phase immunoassay. B, C, D and Q clones producing antibodies against Raja clavata brain gangliosides were obtained. Monoclonal B and C antibodies bound monosialogangliosides. Monoclonal D antibody bound a number of gangliosides but reacted predominantly with GD1a. Monoclonal Q antibody reacted selectively with GQ1c. It is assumed that ganglioside GQ1c is expressed on the melanoma cell surface and may be found only in the early stage of ontogenesis of high vertebrates.     

Reassessing Neotropical angiosperm distribution patterns based on monographic data: a geometric interpolation approach

Monographic data rely on specimens deposited in herbaria and museums, which have been thoroughly revised by experts. However, monographic data have been rarely used to map species richness at large scale, mainly because of the difficulties caused by spatially heterogeneous sampling effort. In this paper we estimate patterns of species richness and narrow endemism, based on monographic data of 4,055 Neotropical angiosperm species. We propose a geometric interpolation method to derive species ranges at a 1° grid resolution. To this we apply an inverse distance-weighted summation scheme to derive maps of species richness and endemism. In the latter we also adjust for heterogeneous sampling effort. Finally, we test the robustness of the interpolated species ranges and derived species richness by applying the same method but using a leave-one-out-cross-validation (LOOCV). The derived map shows four distinct regions of elevated species richness: (1) Central America, (2) the Northern Andes, (3) Amazonia and (4) the Brazilian Atlantic coast (‘Mata Atlantica’). The region with the highest estimated species richness is Amazonia, with Central America following closely behind. Centers of narrow endemism are located over the entire Neotropics, several of them coinciding with regions of elevated species richness. Sampling effort has a minor influence on the interpolation of overall species richness, but it substantially influences the estimation of regions of narrow endemism. Thus, in order to improve maps of narrow endemism and resulting conservation efforts, more collection and identification activity is required.     

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.     

Analysis of three-dimensional ciliary beating by means of high-speed stereomicroscopy.

Results are presented on the analysis of three-dimensional motion of compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia mytilus. Time series of three-dimensional data were obtained by using the anaxial illumination method for simultaneous recording of stereoscopic video images. Data processing involved the following steps: determination of a reference coordinate system based solely on features present in each stereo-pair; tracing of cirral axes in digitized images, conversion to parameter curves by means of least-squares polynomial approximation, conversion of pairs of two-dimensional data to a series of three-dimensional data; correction for distortion due to projective shortening and conversion to a series of polynomial triplets, and analysis of the periodical components of the motion pattern in the frequency domain. Reconstructed beating cycles show typical differences between hyperpolarization-induced ciliary activity and depolarization-induced ciliary activity. Reconstructions of the motion of the basal segment of a cirrus are in agreement with existing data. Analysis of the curvature and torsion of a cirral axis during beating does not reveal any simple pattern of propagated activity within the axoneme. The return stroke may be subdivided into two phases. First, a curvature peak develops proximally. Secondly, a region with increased torsion arises more distally and spreads out in proximal direction. Both curvature and torsion return to minimal values by the beginning of the power stroke.     

Conformational differences between linear alpha (2----8)-linked homosialooligosaccharides and the epitope of the group B meningococcal polysaccharide

The alpha-(2----8)-linked sialic acid oligosaccharides (NeuAc)n exhibit an unusual degree of heterogeneity in the conformation of their linkages. This was diagnosed by observation in their 13C NMR spectra of an equivalent and unique heterogeneity in the chemical shifts of their anomeric carbons and subsequently confirmed by more comprehensive 1H and 13C NMR studies. In these studies both one-dimensional and two-dimensional experiments were carried out on the trisaccharide (NeuAc)3 and colominic acid. In addition to the unambiguous assignment of the signals in the spectra, these experiments demonstrated that both linkages of (NeuAc)3 differed in conformation from each other and from the inner linkages of colominic acid. The NMR data indicate that these conformational differences extend to both terminal disaccharides of oligosaccharides larger than (NeuAc)5, a result that has considerable physical and biological significance. In the context of the group B meningococcal polysaccharide, it provides an explanation for the conformational epitope of the group B meningococcal polysaccharide, which was proposed on the evidence that (NeuAc)10, larger than the optimum size of an antibody site, was the smallest oligosaccharide able to bind to group B polysaccharide specific antibodies. Because the two terminal disaccharides of (NeuAc)10 differ in conformation to its inner residues, the immunologically functional part of (NeuAc)10 resides in its inner six residues. This number of residues is now consistent with the maximum size of an antibody site.