Fracture toughness of mountain gorilla (Gorilla gorilla beringei) food plants

Mountain gorillas, the largest extant primates, subsist almost entirely on plant matter. Moreover, their diet includes a substantial amount of structural material, such as bark and stems, which other primates tend to avoid. Accordingly, the robust masticatory apparatus of gorillas may be adaptive to this presumably tough diet; however, quantitative information on this subject is lacking. In this study the fracture toughness of mountain gorilla foods was examined for the first time. Samples of 44 food plants from Bwindi-Impenetrable National Park (BINP) and Mgahinga Gorilla National Park (MGNP) were tested. These parks are inhabited by two gorilla populations that regarded by some as being distinct at the subspecific taxonomic level. Although food toughness did not differ between the two populations, both diets contained tough items. Tree barks were the toughest food items (varying from 0.23 to 8.2 kJ/m2), followed by shrub barks, pith, and stems. The toughness of leaves and fruit was negligible compared to that of bark. The toughness of bamboo was low in comparison to the toughest food items. Accordingly, the prominent toughness of bark, pith, and stems may be key factors in the evolution of orofacial robusticity in mountain gorillas.     

Free energy surfaces of beta-hairpin and alpha-helical peptides generated by replica exchange molecular dynamics with the AGBNP implicit solvent model

We have studied the potential of mean force of two peptides, one known to adopt a beta-hairpin and the other an alpha-helical conformation in solution. These peptides are, respectively, residues 41-56 of the C-terminus (GEWTYDDATKTFTVTE) of the B1 domain of protein G and the 13 residue C-peptide (KETAAAKFERQHM) of ribonuclease A. Extensive canonical ensemble sampling has been performed using a parallel replica exchange method. The effective potential employed in this work consists of the OPLS all-atom force field (OPLS-AA) and an analytical generalized Born (AGB) implicit solvent model including a novel nonpolar solvation free energy estimator (NP). An additional dielectric screening parameter has been incorporated into the AGBNP model. In the case of the beta-hairpin, the nonpolar solvation free energy estimator provides the necessary effective interactions for the collapse of the hydrophobic core (W43, Y45, F52, and V54), which the more commonly used surface-area-dependent nonpolar model does not provide. For both the beta-hairpin and the alpha-helix, increased dielectric screening reduces the stability of incorrectly formed salt bridges, which tend to disrupt the formation of the hairpin and helix, respectively. The fraction of beta-hairpin and alpha-helix content we obtained using the AGBNP model agrees well with experimental results. The thermodynamic stability of the beta-hairpin from protein G and the alpha-helical C-peptide from ribonuclease A as modeled with the OPLS-AA/AGBNP effective potential reflects the balance between the nonpolar effective potential terms, which drive compaction, and the polar and hydrogen bonding terms, which promote secondary structure formation.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.     

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying

Background

Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze‐drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non‐viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze‐drying and spray‐drying, a one‐step process for producing dry and respirable powders, as methods for preparing non‐viral respiratory gene delivery systems.

Methods

Lipid:polycation:pDNA (LPD) vectors comprising 1,2‐dioleoyl‐3‐trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP‐N1 in 3% lactose solution were either snap‐frozen and lyophilised or spray‐dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence.

Results

Lyophilisation produced large, irregularly shaped particles prior to (mean diameter ～21 µm) and following (mean diameter ～18 µm) coarse grinding. Spray‐drying produced uniformly shaped spherical particles (mean diameter ～4 µm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray‐dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage.

Conclusions

Spray‐drying has utility for producing stable, efficient and potentially respirable non‐viral dry powder systems for respiratory gene delivery. Copyright © 2002 John Wiley & Sons, Ltd.

     

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

Fracture healing as a post-natal developmental process: molecular,spatial, and temporal aspects of its regulation

Fracture healing is a specialized post-natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing, these processes take place in a post-natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the embryonic differentiation and growth of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (1) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular differentiation during repair; (4) the role of three key groups of soluble factors, pro-inflammatory cytokines, the TGF-beta superfamily, and angiogenic factors, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture.     

Are gap junction membrane plaques implicated in intercellular vesicle transfer?

Gap junction channels are concentrated in specialised plaques of plasma membrane where cells are in close apposition. In this communication evidence is provided showing that these specialised regions of membrane also provide a site for vesicular transfer between cells. Vesicle distribution in eye lenses was found to generally reflect the reported distribution of gap junction membrane plaques. In certain areas of the lens gap junction membrane plaques and vesicles could be seen to form combined, complex structures. Ultrastructure of the vesicle and gap junction membrane plaque complexes was consistent with the vesicles moving through membrane plaques from one lens fibre cell to the next. To investigate whether transport of substances was consistent with intercellular vesicle transfer, transport of various markers was investigated. Time course experiments showing the rate of uptake of various markers into the lens did not show dramatic differences for molecules smaller or larger then gap junction pores formed by connexons. While considered as a primary intercellular transport mechanism in the lens, connexon pores were not the sole agent mediating the observed transport. Other reported mechanisms of intercellular transport in the lens can only account for the movement of relatively small molecules. Vesicular transport may therefore be a major form of transport into the outer lens layers for larger molecules. Implicit in these observations is a new hypothesis for intercellular vesicle movement via gap junction membrane plaques. Intercellular vesicle movement could possibly provide a path for large molecules associated with intact vesicles to be transported into the eye lens tissue.     

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.     

Tight Junctions of the Blood–Brain Barrier

1. The blood–brain barrier is essential for the maintainance and regulation of the neural microenvironment. The blood–brain barrier endothelial cells comprise an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. The latter is realized by the tight junctions between the endothelial cells of the brain microvasculature, which are subject of this review. Morphologically, blood–brain barrier-tight junctions are more similar to epithelial tight junctions than to endothelial tight junctions in peripheral blood vessels.2. Although blood–brain barrier-tight junctions share many characteristics with epithelial tight junctions, there are also essential differences. However, in contrast to tight junctions in epithelial systems, structural and functional characteristics of tight junctions in endothelial cells are highly sensitive to ambient factors.3. Many ubiquitous molecular constituents of tight junctions have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Signaling pathways involved in tight junction regulation comprise, among others, G-proteins, serine, threonine, and tyrosine kinases, extra- and intracellular calcium levels, cAMP levels, proteases, and TNF. Common to most of these pathways is the modulation of cytoskeletal elements which may define blood–brain barrier characteristics. Additionally, cross-talk between components of the tight junction– and the cadherin–catenin system suggests a close functional interdependence of the two cell–cell contact systems.4. Recent studies were able to elucidate crucial aspects of the molecular basis of tight junction regulation. An integration of new results into previous morphological work is the central intention of this review.