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PubMed1996

Bioprocessing of sweet sorghum with in situ-produced enzymes.

Enzyme-assisted ensiling (ENLAC), using in situ-produced enzymes from Gliocladium sp. TUB-F-498, preserved 80% of the sugar content of sweet sorghum, and facilitated its extraction by countercurrent diffusion. The in situ enzyme was produced on the extracted sweet sorghum pulp by an 8-d solid substrate fermentation (SSF) with a yield of 4.6 cellulase and 400 IU/g dry wt xylanase. Two percent of the fermented substrate had cellulase and xylanase levels equivalent or superior to levels found in the commercial enzymes Celluclast and Viscozyme Novo at the 0.025% application level in ENLAC. The in situ-production of enzymes on recyclable substrates may reduce bioprocessing costs significantly. In this ENLAC process, the cost of the in situ enzymes is estimated to be about $0.12/MT substrate, compared to $9.5/metric ton (MT) for the commercial enzymes, a cost reduction of nearly 80-fold.

Biotechnology↗
PubMed1990

Three-dimensional structure of the mammalian cytoplasmic ribosome.

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.

Animals↗
PubMed1984

Plasma ascorbate concentrations and blood cell dehydroascorbate transport in patients with diabetes mellitus.

Rates of dehydroascorbate uptake by blood granulocytes and mononuclear cells are slower, and plasma ascorbate concentrations are lower, among persons with diabetes mellitus than in nondiabetic subjects. These measurements do not correlate with one another or with simultaneously measured plasma glucose or glycosylated hemoglobin; they do not differ with type of diabetes or mode of treatment. In those diabetic granulocytes that exhibit slow dehydroascorbate uptake, maximal velocity (Vmax) transport rates for dehydroascorbate, 2-deoxyglucose, and 3-O-methylglucose are decreased, each to the same degree, while Km values for transport of these ligands are not different from those observed in nondiabetic cells. Since diffusion of these ligands is facilitated by a common transporter, these observations may reflect decreased numbers of glucose transporters in the plasma membranes of some diabetic leukocytes.

3-O-Methylglucose↗
PubMed1985

Comparison of percutaneous absorption of fragrances by humans and monkeys.

The percutaneous absorption of two cosmetic fragrance materials, safrole and cinnamyl anthranilate, as well as of cinnamic alcohol and cinnamic acid, has been measured at occluded and non-occluded application sites. Absorption values were determined in the rhesus monkey in vivo. Absorption through human skin was measured by using excised skin in diffusion cells. Because of the insolubility in water of safrole and cinnamyl anthranilate, a nonionic surfactant solution (6% oleth 20) was used in the receptor chamber of the diffusion cell in order to facilitate the partitioning of the compounds from the skin into the receptor fluid. The relative volatility of the compounds was determined in order to aid in the interpretation of the absorption results. The greatest difference between in vivo and in vitro absorption values occurred with safrole, which was the least well absorbed and the most volatile compound. Cinnamic acid absorption through non-occluded human skin (17.8 +/- 4.9%, mean +/- SEM) was significantly lower than through monkey skin (38.6 +/- 8.3%). The values for absorption through human and monkey skin did not differ significantly for cinnamyl anthranilate (24.0 +/- 5.1% v. 26.1 +/- 2.3%) or cinnamic alcohol (33.9 +/- 7.3% v. 25.4 +/- 4.4%). Occlusion of the skin resulted in greater permeation of all of the compounds; a significant difference in permeability between the two types of skin occurred only with safrole. The fragrances were absorbed well, but their volatility must be considered in a toxicity evaluation. There was reasonable agreement between the values obtained from the studies of the human skin in vitro and the monkey skin in vivo.

Animals↗
PubMedDate not supplied

Effects of Ginkgo biloba extract on glucose transport and glycogen synthesis of cultured smooth muscle cells from pig aorta.

We have examined the effect of an extract of Ginkgo biloba (Gbe) on glucose uptake and on glycogen synthesis in cultured smooth muscle cells (SMC) from pig aorta. Initial rates of glucose transport were determined by measurements of 2-deoxy-D-glucose (2-DG) uptake. From kinetic analyses apparent KM and Vmax values of facilitated glucose transport in cultured SMC were evaluated at 2.2 mM and 9.1 nmol/min/10(6) cells respectively. Gbe stimulated glucose transport in a dose-dependent manner; the maximum effect was reached at a Gbe concentration of 0.25 micrograms/ml and represented an increase of 35 +/- 4% above basal activity. This stimulation mainly occurred on facilitated glucose transport. The passive diffusion measured when cells were treated with cytochalasin B represented 15 +/- 3% of glucose total transport activity either in the absence or the presence of Gbe. The effect of Gbe on glycogen synthesis in cultured SMC was then tested by the incorporation of U14C-glucose into cellular glycogen. This process was enhanced by Gbe, the maximal effect was observed at a Gbe concentration of 0.25 micrograms/ml, and represented a 41 +r4% increase above basal activity. These data argue for a direct effect of Gbe upon glucose transport and glucose utilization in cultured SMC thus allowing a better nutriment disposal in the vascular wall.

Animals↗
PubMed2026

Divergent evolutionary strategies in spider venoms: A comparative proteomic profiling of four sympatric species from Yunnan.

Spider venoms comprise complex cocktails of bioactive molecules evolved for predation and defense, representing a valuable resource for biological research and pharmaceutical discovery. In this study, we performed a systematic analysis of venom gland extracts from four common spider species indigenous to Yunnan, China: Agelena limbata, Hippasa lycosina, Lycosa grahami, and Sinopoda pengi. Using an integrated transcriptomic and proteomic targeted profiling approach, we successfully annotated 141 distinct toxins. Comparative analysis revealed significant interspecific heterogeneity, suggesting distinct evolutionary trajectories and "weapon system economics." Both A. limbata and L. grahami exhibited a "peptide-dominant" profile anchored by neurotoxic peptides and isomerases, optimized for rapid chemical paralysis. In contrast, S. pengi displayed a distinct "protein-dominant" signature enriched with high-molecular-weight enzymes and CAP superfamily proteins, likely functioning to facilitate tissue degradation and toxin diffusion. Occupying an intermediate position, H. lycosina demonstrated a hybrid composition. These findings suggest that although these species share the same geographical range, their venom systems have undergone divergent evolutionary adaptations driven by specific ecological niches and hunting strategies. This study represents the first systematic proteomic characterization of these venom components, providing a valuable reservoir of molecular candidates while highlighting the bioinformatic nuances of analyzing whole-gland homogenates.

Animals↗
PubMed1998

Viewpoint: the core and matrix of thalamic organization.

The integration of the whole cerebral cortex and thalamus during forebrain activities that underlie different states of consciousness, requires pathways for the dispersion of thalamic activity across many cortical areas. Past theories have relied on the intralaminar nuclei as the sources of diffuse thalamocortical projections that could facilitate spread of activity across the cortex. A case is made for the presence of a matrix of superficially-projecting cells, not confined to the intralaminar nuclei but extending throughout the whole thalamus. These cells are distinguished by immunoreactivity for the calcium-binding protein, D28K calbindin, are found in all thalamic nuclei of primates and have increased numbers in some nuclei. They project to superficial layers of the cerebral cortex over relatively wide areas, unconstrained by architectonic boundaries. They generally receive subcortical inputs that lack the topographic order and physiological precision of the principal sensory pathways. Superimposed upon the matrix in certain nuclei only, is a core of cells distinguished by immunoreactivity for another calcium-binding protein, parvalbumin, These project in highly ordered fashion to middle layers of the cortex in an area-specific manner. They are innervated by subcortical inputs that are topographically precise and have readily identifiable physiological properties. The parvalbumin cells form the basis for sensory and other inputs that are to be used as a basis for perception. The calbindin cells, especially when recruited by corticothalamic connections, can form a basis for the engagement of multiple cortical areas and thalamic nuclei that is essential for the binding of multiple aspects of sensory experience into a single framework of consciousness.

Animals↗
PubMed2003

Muscle metabolism and growth in Antarctic fishes (suborder Notothenioidei): evolution in a cold environment.

The radiation of notothenioid fishes (order Perciformes) in the Southern Ocean provides a model system for investigating evolution and adaptation to a low temperature environment. The Notothenioid fishes comprising eight families, 43 genera and 122 species dominate the fish fauna in Antarctica. The diversification of the clade probably began 15-20 million years ago after the formation of the Antarctic Polar Front. The radiation was, therefore, associated with climatic cooling down to the present day temperature of -1.86 degrees C. Origins and Evolution of the Antarctic Biota Geological Society Special Publication No. 47, Geological Society of London. pp. 253-268). The success of the group has been closely linked with the evolution of glycopeptide and peptide antifreezes, which are amongst the most abundant proteins in blood and interstitial fluid. The radiation of the clade has been associated with disaptation (evolutionary loss of function) and recovery. For example, it is thought that the icefishes (Channichyidae) lost haemoglobin through a single mutational event leading to the deletion of the entire beta-globin gene and the 5' end of the linked alpha-globin gene, resulting in compensatory adaptations of the cardiovascular system. Phylogenetically based statistical methods also indicate a progressive and dramatic reduction in the number of skeletal muscle fibres (FN(max)) at the end of the recruitment phase of growth in basal compared to derived families. The reduction in FN(max) is associated with a compensatory increase in the maximum fibre diameter, which can reach 100 microm in slow and 600 microm in fast muscle fibres. At -1 to 0 degrees C, the oxygen consumption of isolated mitochondria per mg mitochondrial protein shows no evidence of up-regulation relative to mitochondria from temperate and tropical Perciform fishes. The mitochondria content of slow muscle fibres in Antarctic notothenioids is towards the upper end of the range reported for teleosts with similar lifestyles, reaching 50% in Channichthyids. High mitochondrial densities facilitate ATP production and oxygen diffusion through the membrane lipid compartment of the fibre. Modelling studies suggest that adequate oxygen flux in the large diameter muscle fibres of notothenioids is possible because of the reduced metabolic demand and enhanced solubility of oxygen associated with low temperature. At the whole animal level size-corrected resting metabolic rate fits on the same temperature relationship as for Perciformes from warmer climates. It seems likely that the additional energetic costs associated with antifreeze synthesis and high mitochondrial densities are compensated for by reductions in other energy requiring processes: a hypothesis that could be tested with detailed energy budget studies. One plausible candidate is a reduction in membrane leak pathways linked to the loss of muscle fibres, which would serve to minimise the cost of maintaining ionic gradients.

Animals↗
PubMed1988

When does information change practitioners' behavior?

Programs that disseminate information to health care practitioners often do so partly to encourage appropriate changes in practice. However, merely providing information is seldom enough to accomplish such changes. If information transfer programs are to influence practice, they must be designed to maximize the conditions facilitating change. Reliance on a diffusion model for thinking about how information reaches practitioners has led researchers to over-emphasize the importance of exposure to information and ignore other factors that determine whether change will occur, such as practitioners' motivation to change, the context in which clinical decisions are made, and how information is presented. The fact that successful dissemination will not necessarily produce change also has implications for how information transfer programs should be monitored and evaluated.

Attitude of Health Personnel↗
PubMed2005

Effect of chemical speciation on toxicity of mercury to Escherichia coli biofilms and planktonic cells.

While it is known that microbial uptake of mercury (Hg) by planktonic cultures is influenced by the extracellular speciation of mercury in aquatic systems, Hg uptake in biofilm cultures is understudied. We compared the importance of Hg(II) speciation in toxicity to both planktonic and biofilm cultures of the Gram-negative bacterium Escherichia coli 055. Variable chloride chemistry experiments were carried out to modify mercury speciation. Biofilms were observed to be more resistant to Hg than planktonic cells. In both planktonic and biofilm cultures, the toxicity of Hg increased and then decreased along the chloride gradient. The percent reduction in cell viability was linearly related to the concentration of HgCl2(0) when Hg-chloro complexes dominated the speciation, consistent with a passive diffusion model. However, toxicity to both planktonic cells and biofilms at low salinities could not be explained by passive diffusion alone, which suggests that microbial uptake of Hg in both planktonic cells and biofilms may occur by both passive diffusion of neutral species and facilitated uptake. The relationship between toxicity and chloride concentration was similar in the presence and absence of a biofilm, indicating that the presence of the biofilm does not drastically change the relative availability of the dominant mercury species.

Biofilms↗
PubMed1997

Membrane transport in hepatic clearance of drugs. I: Extended hepatic clearance models incorporating concentration-dependent transport and elimination processes.

PURPOSE: The objective of the present study was to develop hepatic clearance models which incorporate a unidirectional carrier-mediated uptake and bidirectional diffusional transport processes for drug transport in the sinusoidal membrane of hepatocytes as well as nonlinear intrinsic elimination. METHODS: Two models were derived which view the liver as two separate compartments, i.e., sinusoid and hepatocyte. Model I assumes the instantaneous complete mixing of drugs within each compartment (similar to that of the "well-stirred" model), while model II assumes that the drug concentrations in both compartments decrease progressively in the direction of the hepatic blood flow path (similar to that of the "parallel-tube" model). Computer simulations were performed using a range of steady-state infusion rates for a substrate, while varying the Vmax (capacity) and Km (Michaelis-Menten constant) for the carrier-mediated uptake process, the diffusional clearance, the Vmax and Km for the intrinsic elimination process, blood flow and protein binding. RESULTS: Simulations in which Vmax and Km for the sinusoidal membrane transporter and the diffusional clearance were varied, demonstrated that these membrane transport processes could affect the clearance of drugs to a significant extent in both models. The estimates for clearance of substrates with the same pharmacokinetic parameters are always lower in model I than in model II, although the quantitative differences in parameter estimates between models varied, depending on the steady state infusion rates. CONCLUSIONS: These more general hepatic clearance models will be most useful for describing the hepatic clearance of hydrophilic compounds, such as organic anions or cations, which exhibit facilitated uptake and limited membrane diffusion in hepatocytes.

Biological Transport↗
PubMed1992

Protein folding funnels: a kinetic approach to the sequence-structure relationship.

A lattice model of protein folding is developed to distinguish between amino acid sequences that do and do not fold into unique conformations. Although Monte Carlo simulations provide insights into the long-time processes involved in protein folding, these simulations cannot systematically chart the conformational energy surface that enables folding. By assuming that protein folding occurs after chain collapse, a kinetic map of important pathways on this surface is constructed through the use of an analytical theory of probability flow. Convergent kinetic pathways, or "folding funnels," guide folding to a unique, stable, native conformation. Solution of the probability flow equations is facilitated by limiting treatment to diffusion between geometrically similar collapsed conformers. Similarity is measured in terms of a reconfigurational distance. Two specific amino acid sequences are deemed foldable and nonfoldable because one gives rise to a single, large folding funnel leading to a native conformation and the other has multiple pathways leading to several stable conformers. Monte Carlo simulations demonstrate that folding funnel calculations accurately predict the fact of and the pathways involved in folding-specific sequences. The existence of folding funnels for specific sequences suggests that geometrically related families of stable, collapsed conformers fulfill kinetic and thermodynamic requirements of protein folding.

Algorithms↗
PubMed2006

Uses and abuses of EIDORS: an extensible software base for EIT.

EIDORS is an open source software suite for image reconstruction in electrical impedance tomography and diffuse optical tomography, designed to facilitate collaboration, testing and new research in these fields. This paper describes recent work to redesign the software structure in order to simplify its use and provide a uniform interface, permitting easier modification and customization. We describe the key features of this software, followed by examples of its use. One general issue with inverse problem software is the difficulty of correctly implementing algorithms and the consequent ease with which subtle numerical bugs can be inadvertently introduced. EIDORS helps with this issue, by allowing sharing and reuse of well-documented and debugged software. On the other hand, since EIDORS is designed to facilitate use by non-specialists, its use may inadvertently result in such numerical errors. In order to address this issue, we develop a list of ways in which such errors with inverse problems (which we refer to as 'cheats') may occur. Our hope is that such an overview may assist authors of software to avoid such implementation issues.

Algorithms↗
PubMed2002

Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes.

The mechanism by which macromolecules are translocated through the nuclear pore complex (NPC) is little understood. However, recent measurements of nuclear transport in permeabilized cells showed that molecules binding to phenylalanine-glycine-rich repeats (FG repeats) in NPC proteins were translocated much faster through the NPC than molecules not interacting with FG repeats. We have studied that substrate preference of the NPC in isolated oocyte nuclei and purified nuclear envelopes by optical single transporter recording. NTF2, the transport receptor of RanGDP, was exported approximately 30 times faster than green fluorescent protein, an inert molecule of approximately the same size. The data confirm that restricted diffusion of inert molecules and facilitated transport of FG-repeat binding proteins are basic types of translocation through the NPC, demonstrating that the functional integrity of the NPC can be conserved in isolated nuclei and nuclear envelopes and thus opening new avenues to the analysis of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗
PubMed2001

Mechanisms involved in the intestinal digestion and absorption of dietary vitamin A.

Dietary retinyl esters are hydrolyzed in the intestine by the pancreatic enzyme, pancreatic triglyceride lipase (PTL), and intestinal brush border enzyme, phospholipase B. Recent work on the carboxylester lipase (CEL) knockout mouse suggests that CEL may not be involved in dietary retinyl ester digestion. The possible roles of the pancreatic lipase-related proteins (PLRP) 1 and 2 and other enzymes require further investigation. Unesterified retinol is taken up by the enterocytes, perhaps involving both diffusion and protein-mediated facilitated transport. Once in the cell, retinol is complexed with cellular retinol-binding protein type 2 (CRBP2) and the complex serves as a substrate for reesterification of the retinol by the enzyme lecithin:retinol acyltransferase (LRAT). Retinol not bound to CRBP2 is esterified by acyl-CoA acyltransferase (ARAT). The retinyl esters are incorporated into chylomicrons, intestinal lipoproteins that transport other dietary lipids such as triglycerides, phospholipids, and cholesterol. Chylomicrons containing newly absorbed retinyl esters are then secreted into the lymph.

Animals↗
PubMed1992

Reactive production of extracellular matrix (ECM) by corneal endothelial cells.

Results of experimental and histopathological studies of reactive production of extracellular matrix by corneal endothelial cells are summarized and evaluated. In anterior synechiae, epithelial ingrowth and a chronic corneal ulcer extracellular matrix was produced not only toward Descemet's membrane but also on the normally free endothelial cell surface. In some cases of anterior synechiae and cystic epithelial ingrowth, after partial separation of the new layer of extracellular matrix, recovery and regeneration of endothelial cells was possible. In diffuse epithelial invasion, extracellular matrix facilitated at the beginning the progression of epithelium over endothelium, putatively serving as a substrate; however, in longer-standing invasions endothelial cells degenerated and died. Reactive production of extracellular matrix may represent a defensive mechanism of endothelium threatened by foreign cells, tissues or materials.

Animals↗
PubMed1996

Vertical Migration in the Sediment-Dwelling Sulfur Bacteria Thioploca spp. in Overcoming Diffusion Limitations.

In order to investigate the environmental requirements of the filamentous sulfur bacteria Thioploca spp., we tested the chemotactic responses of these sedimentary microorganisms to changes in oxygen, nitrate, and sulfide concentrations. A sediment core with a Thioploca mat, retrieved from the oxygen-minimum zone on the Chilean shelf, was incubated in a recirculating flume. The addition of 25 (mu)mol of nitrate per liter to the seawater flow induced the ascent of the Thioploca trichomes (length, up to 70 mm) in their mostly vertically oriented gelatinous sheaths. The upper ends of the filaments penetrated the sediment surface and protruded 1 to 3 mm into the flowing water before they bent downstream. By penetrating the diffusive boundary layer, Thioploca spp. facilitate efficient nitrate uptake in exposed trichome sections that are up to 30 mm long. The cumulative length of exposed filaments per square centimeter of sediment surface was up to 92 cm, with a total exposed trichome surface area of 1 cm(sup2). The positive reaction to nitrate overruled a negative response to oxygen, indicating that nitrate is the principal electron acceptor used by Thioploca spp. in the anoxic environment; 10-fold increases in nitrate fluxes after massive emergence of filaments strengthened this hypothesis. A positive chemotactic response to sulfide concentrations of less than 100 (mu)mol liter(sup-1) counteracted the attraction to nitrate and, along with phobic reactions to oxygen and higher sulfide concentrations, controlled the vertical movement of the trichomes. We suggest that the success of Thioploca spp. on the Chilean shelf is based on the ability of these organisms to shuttle between the nitrate-rich boundary layer and the sulfidic sediment strata.

Journal Article↗
PubMed2001

Enhancement of beta-sitosterol transformation in Mycobacterium vaccae with increased cell wall permeability.

Mycobacterium vaccae exposed to compounds which are known to disorganise the cell wall composition and architecture (protamine, glycine) showed increased specific activity in beta-sitosterol biotransformation to androstene derivatives, intennediates in the production of most medical steroids. GC/MS analysis of free lipid fatty acids revealed higher content of unsaturated compounds, mainly C16:1 and C18:1 in protamine- and glycine-treated cells than that in control cells, which seems to change the permeability features of the cell wall barrier, facilitating hydrophobic beta-sitosterol diffusion.

Androstadienes↗