Simulations of molecular diffusion in lattices of cells: insights for NMR of red blood cells

The pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiment, conducted on a suspension of red blood cells (RBC) in a strong magnetic field yields a q-space plot consisting of a series of maxima and minima. This is mathematically analogous to a classical optical diffraction pattern. The method provides a noninvasive and novel means of characterizing cell suspensions that is sensitive to changes in cell shape and packing density. The positions of the features in a q-space plot characterize the rate of exchange across the membrane, cell dimensions, and packing density. A diffusion tensor, containing information regarding the diffusion anisotropy of the system, can also be derived from the PGSE NMR data. In this study, we carried out Monte Carlo simulations of diffusion in suspensions of "virtual" cells that had either biconcave disc (as in RBC) or oblate spheroid geometry. The simulations were performed in a PGSE NMR context thus enabling predictions of q-space and diffusion tensor data. The simulated data were compared with those from real PGSE NMR diffusion experiments on RBC suspensions that had a range of hematocrit values. Methods that facilitate the processing of q-space data were also developed.     

Mean residence time of molecules diffusing in a cell bounded by a semi-permeable membrane: Monte Carlo simulations and an expression relating membrane transition probability to permeability

 The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn²⁺ and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P _d. This relationship was derived and used to show that the mean residence time for a water molecule is determined by P _d when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value. Received: 7 January 2000 / Revised version: 4 April 2000 / Accepted: 4 April 2000     

Proteomics of Mycoplasma genitalium: identification and characterization of unannotated and atypical proteins in a small model genome

We present the results of a comprehensive analysis of the proteome of Mycoplasma genitalium (MG), the smallest autonomously replicating organism that has been completely sequenced. Our aim was to identify and characterize all soluble proteins in MG that are structurally and functionally uncharacterized. We were particularly interested in identifying proteins that differed significantly from typical globular proteins, for example, proteins which are unstructured in the absence of a ‘partner’ molecule or those that exhibit unusual thermodynamic properties. This work is complementary to other structural genomics projects whose primary aim is to determine the three-dimensional structures of proteins with unknown folds. We have identified all the full-length open reading frames (ORFs) in MG that have no homologs of known structure and are of unknown function. Twenty-five of the total 483 ORFs fall into this category and we have expressed, purified and characterized 11 of them. We have used circular dichroism (CD) to rapidly investigate their biophysical properties. Our studies reveal that these proteins have a wide range of structures varying from highly helical to partially structured to unfolded or random coil. They also display a variety of thermodynamic properties ranging from cooperative unfolding to no detectable unfolding upon thermal denaturation. Several of these proteins are highly conserved from mycoplasma to man. Further information about target selection and CD results is available at http://bioinfo.mbb.yale.edu/genome     

Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene

The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.     

A synthetic peptide ligand of neural cell adhesion molecule (NCAM) IgI domain prevents NCAM internalization and disrupts passive avoidance learning

The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.     

Carnivora from the Plio-Pleistocene hominin site of Drimolen, Gauteng, South Africa

Drimolen is one of the newest and most productive hominin sites in South Africa, and is dated on faunal grounds between 2.0 Ma to 1.5 Ma. This paper provides the first overview of the Carnivora from Drimolen, updating the previously published preliminary faunal list, and describing all currently prepared craniodental and postcranial material. The Drimolen specimens are described in comparison with other modern and fossil South African carnivore material. The carnivores cover a range of taxa including hyaenids, felids, canids and herpestids. Most notable amongst these are the sabretooth Dinofelis aff. piveteaui craniodental and postcranial remains, which are described in detail, and a Chasmaporthetes nitidula cranium. The genus Chasmaporthetes is found at three other sites in the area - Sterkfontein, Swartkrans and Coopers D. There are two models for the geographic origin of Dinofelis piveteaui, in that it may have arisen in either eastern or southern Africa. These possibilities are discussed in the light of the new South African Dinofelis material, as the Drimolen material appears to represent a more primitive form with affinities with D. piveteaui. Fossil leopard material from Kromdraai B and Drimolen is also discussed, as the metapodia assigned to P. pardus from these two sites are very small, but lie within the variation of modern leopards. Such size differences in fossil postcrania may have implications for the niches that these animals may have occupied in the past.     

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.     

New materials from proteins and peptides

In this review we highlight recent accomplishments in the design of materials from proteins and peptides. Examples include hydrogels made from aggregating designed β-hairpin peptides, whose physical properties respond to small changes in the amino acid composition of the peptide; materials that combine different segments of natural elastomeric proteins - such as elastin, resilin, silk fibroin whose bulk properties are dictated in unanticipated ways by their composition; and hydrogels formed by strings or arrays of protein modules, which are cross-linked by multivalent versions of their peptide ligands, and which may exhibit exquisite stimuli-responsive behavior. The suitability of the unique properties of such new materials for practical applications is also considered.