Biochemical Flux Research Articles

Genome-scale community modelling elucidates the metabolic interaction in Indian type-2 diabetic gut microbiota

Type-2 diabetes (T2D) is a rapidly growing multifactorial metabolic disorder that induces the onset of various diseases in the human body. The compositional and metabolic shift of the gut microbiota is a crucial factor behind T2D. Hence, gaining insight into the metabolic profile of the gut microbiota is essential for revealing their role in regulating the metabolism of T2D patients. Here, we have focused on the genome-scale community metabolic model reconstruction of crucial T2D-associated gut microbes. The model-based analysis of biochemical flux in T2D and healthy gut conditions showed distinct biochemical signatures and diverse metabolic interactions in the microbial community. The metabolic interactions encompass cross-feeding of short-chain fatty acids, amino acids, and vitamins among individual microbes within the community. In T2D conditions, a reduction in the metabolic flux of acetate, butyrate, vitamin B5, and bicarbonate was observed in the microbial community model, which can impact carbohydrate metabolism. The decline in butyrate levels is correlated with both insulin resistance and diminished glucose metabolism in T2D patients. Compared to the healthy gut, an overall reduction in glucose consumption and SCFA production flux was estimated in the T2D gut environment. Moreover, the decreased consumption profiles of branch chain amino acids (BCAAs) and aromatic amino acids (AAAs) in the T2D gut microbiota can be a distinct biomarker for T2D. Hence, the flux-level analysis of the microbial community model can provide insights into the metabolic reprogramming in diabetic gut microbiomes, which may be helpful in personalized therapeutics and diet design against T2D.

Reconstruction of a Genome-Scale Metabolic Model of Scenedesmus obliquus and Its Application for Lipid Production under Three Trophic Modes.

Green microalgae have emerged as beneficial feedstocks for biofuel production. A systems-level understanding of the biochemical network is needed to harness the microalgal metabolic capacity for bioproduction. Genome-scale metabolic modeling (GEM) showed immense potential in rational metabolic engineering, utilizing biochemical flux distribution analysis. Here, we report the first GEM for the green microalga, Scenedesmus obliquus (iAR632), a promising biodiesel feedstock with high lipid-storing capability. iAR632 comprises 1467 reactions, 734 metabolites, and 632 genes distributed among 7 compartments. The model was optimized under three different trophic modes of microalgal cultivation, i.e., autotrophy, mixotrophy, and heterotrophy. The robustness of the reconstructed network was confirmed by analyzing its sensitivity to the biomass components. Pathway-level flux profiles were analyzed, and significant flux space expansion was noticed majorly in reactions associated with lipid biosynthesis. In agreement with the experimental observation, iAR632 predicted about 3.8-fold increased biomass and almost 4-fold higher lipid under mixotrophy than the other trophic modes. Thus, the assessment of the condition-specific metabolic flux distribution of iAR632 suggested that mixotrophy is the preferred cultivation condition for improved microalgal growth and lipid production. Overall, the reconstructed GEM and subsequent analyses will provide a systematic framework for developing model-driven strategies to improve microalgal bioproduction.

Moved by Another Life: Altered Sentience and Historical Poiesis in the Peyote Craze

Abstract This article follows the transsensory pathways opened by peyote, a mood-altering entheogen, as it diffused among Native peoples living under and along the edges of colonial occupation around the turn of the twentieth century. It traces that movement through the pulsions of temporal and sensorial animacy created in the episodic narration of The Autobiography of a Winnebago Indian (1920), the life story of a Ho-Chunk man named Sam Blowsnake. Apprehended by settler governance as a “craze,” anti-assimilationist currents of the Peyote movement met with intensifying attempts to constrain the biochemical flux of “intoxication” through Native bodies. Modern campaigns against “peyote worship” mounted by government agents, progressive reformers, and sensationalist press in the early twentieth century exploited scripts of revulsion and prurient fascination to target the border-crossing “mind poison” for prohibition. Just beneath the scenes of psychotoxicity and sexual disorder projected by the antinarcotic imaginary of the craze, this article posits, there ran an ongoing struggle over the restrictive capacitation of property and personhood within the settler-capitalist regime of allotment. Against the norms enforced by that policy, in peyote meetings the alteration of sentience could unbind the day-to-day reproduction of property-bearing personhood. Lines of collective transport opening from the passage of ecstasy thus composed a historical moment in refusal of allotment drives for schizochronic individuation.

Spectral Characterization of Stem Cell-Derived Myelination within the Injured Adult PNS Using the Solvatochromic Dye Nile Red.

Background: Myelin is an essential component of the peripheral and central nervous system, enabling fast axonal conduction and supporting axonal integrity; limited tools exist for analysis of myelin composition in-vivo. Objective: To demonstrate that the photophysical properties of myelin-incorporated solvatochromic dyes can be exploited to probe the biochemical composition of living peripheral nerve myelin at high spatial resolution. Methods: Using the myelin-incorporated fluorescent dye Nile Red we sequentially analyzed the spectral characteristics of remyelinating myelin membranes both in-vitro and in-vivo, including in living rats. Results: We demonstrated a consistent bi-phasic evolution of emission spectra during early remyelination, and visually report the reliable biochemical flux of myelin membrane composition in-vitro and in-vivo. Conclusions: Solvatochromic spectroscopy enables the analysis of myelin membrane maturity during remyelination, and can be performed in-vivo. As the formation of myelin during early-to-late remyelination likely incorporates fluctuating fractions of lipophilic components and changes in lateral membrane mobility, we propose that our spectrochemical data reflects the observation of these biochemical processes.

Key Concepts Surrounding Studies of Stable Isotope-Resolved Metabolomics.

"Omics"-based analyses are widely used in numerous areas of research, advances in instrumentation (both hardware and software) allow investigators to collect a wealth of data and therein characterize metabolic systems. Although analyses generally examine differences in absolute or relative (fold-) changes in concentrations, the ability to extract mechanistic insight would benefit from the use of isotopic tracers. Herein, we discuss important concepts that should be considered when stable isotope tracers are used to capture biochemical flux. Special attention is placed on in vivo systems, however, many of the general ideas have immediate impact on studies in cellular models or isolated-perfused tissues. While it is somewhat trivial to administer labeled precursor molecules and measure the enrichment of downstream products, the ability to make correct interpretations can be challenging. We will outline several critical factors that may influence choices when developing and/or applying a stable isotope tracer method. For example, is there a "best" tracer for a given study? How do I administer a tracer? When do I collect my sample(s)? While these questions may seem straightforward, we will present scenarios that can have dramatic effects on conclusions surrounding apparent rates of metabolic activity.

The combined effect of wall shear stress topology and magnitude on cardiovascular mass transport

Cardiovascular disease typically initiates at the vessel wall where near-wall transport of certain biochemicals influences disease initiation and progression. Wall shear stress (WSS) influences these transport processes in a complex manner. WSS magnitude and the shear force exerted on the endothelial cells determine the biochemical flux at the vessel wall. In addition, it has been recently shown that Lagrangian WSS structures (topological features) influence near-wall transport in high Schmidt and Peclet numbers. In this study, the influence of WSS topology and magnitude on surface concentration patterns in shear-dependent biochemical transport problems was explored in a coronary artery stenosis and a carotid artery model. Shear-enhanced and shear-reduced biochemical flux boundary conditions were defined at the wall and surface concentration patterns were obtained by solving the advection-diffusion-reaction equation using the finite element method. Surface concentration patterns were demonstrated to depend on a competition between WSS topology and magnitude. A threshold point was identified where WSS topology determined surface concentration patterns for flux equations closer to homogeneous flux, whereas WSS magnitude dominated surface concentration once the wall flux was sufficiently heterogeneous and strongly dependent on shear stress. Finally, nitric oxide (NO) transport was investigated as an example of an important biochemical with shear-enhanced flux at the vessel wall. It was shown that NO transport was close to the identified threshold where WSS topology and magnitude both influenced surface concentration. This study shows that WSS could potentially be used as a powerful parameter to predict qualitative surface concentration patterns without the need to solve numerically challenging cardiovascular mass transport problems.

Phosphorus and nitrogen starvation reveal life‐cycle specific responses in the metabolome of Emiliania huxleyi (Haptophyta)

AbstractThe coccolithophore Emiliania huxleyi is a microalga with biogeochemical and biotechnological relevance, due to its high abundance in the ocean and its ability to form intricate calcium carbonate structures. Depletion of macronutrients in oceanic waters is very common and will likely enhance with advancing climate change. We present the first comprehensive metabolome study analyzing the effect of phosphorus (P) and nitrogen (N) starvation on the diploid and haploid life‐cycle stage, applying various metabolome analysis methods to gain new insights in intracellular mechanisms to cope with nutrient starvation. P‐starvation led to an accumulation of many generic and especially N‐rich metabolites, including lipids, osmolytes, and pigments. This suggests that P‐starvation primarily arrests cell‐cycling due to lacking P for nucleic acid synthesis, but that enzymatic functionality is widely preserved. Also, the de‐epoxidation ratio of the xanthophyll cycle was upregulated in the diploid stage under P‐starvation, indicating increased nonphotochemical quenching, a response typically observed under high light stress. In contrast, N‐starvation resulted in a decrease of most central metabolites, also P‐containing ones, especially in the diploid stage, indicating that most enzymatic functionality ceased. The two investigated nutrient starvation conditions caused significantly different responses, contrary to previous assumptions derived from transcriptomic studies. Data highlight that instantaneous biochemical flux is a more dominant driver of the metabolome than the transcriptomically rearranged pathway patterns. Due to the fundamental nature of the observed responses it may be speculated that microalgae with similar nutrient requirements can cope better with P‐starvation than with N‐starvation.

Enhancing Studies of Pharmacodynamic Mechanisms via Measurements of Metabolic Flux: Fundamental Concepts and Guiding Principles for Using Stable Isotope Tracers.

Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.

Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase

Motivations for the hierarchical assembly of protein complexes are diverse spanning biosensing, biomedical and bioreactor applications. The assembly processes should be simple, scalable, versatile, and biologically benign to minimize loss of component parts. A “plug and play” methodology comprising a generic linking apparatus may enable rapid design and optimization. One application that desires these qualities is metabolon construction wherein multiple enzymes are organized in defined pathways to mediate biochemical flux. Here, we propose a modular design by incorporation of crosslinking-compliant amino acid tags comprised of lysine or glutamine residues at the N- or C-termini of the to-be-assembled proteins. These amino acid tags enable covalent crosslinking using microbial transglutaminase (mTG). Modularity is demonstrated where stoichiometries and relative positions of enzymes and other functional proteins are altered. Construction of multifunctional complexes is demonstrated by crosslinking domains of different function and origin. Namely, we built a two-subunit quorum sensing (QS) biosynthetic metabolon on solid supports and altered stoichiometries of the limiting constituents to increase the overall rate of reaction. To display functionality beyond biosynthesis, we constructed a molecular communication ‘device’ (antibody binding Protein G–QS complex) to target bacterial cells and demonstrated tailored QS responses among targeted bacteria. We propose that this approach, solid phase mTG-mediated linkage of biological components, can be used for assembly within many environments including microreactors or lab-on-a-chip systems. Because the methodology is general, we envision construction of multi-functional protein complexes in a ‘plug and play’ fashion for a variety of biosensing and synthetic biology applications.

Abstract A14: 13C metabolomic and fluxomic study of human B-cell lymphoma metabolic network: Quantifying glutaminolysis and reductive carboxylation

Abstract Intracellular 13C LC-MS and MRS isotopomer data coupled with metabolic modeling allows quantitative determination of metabolic fluxes and other metabolic parameters in vivo/in situ (1). Utilizing these methods, we have determined how an mTOR inhibitor, rapamycin, alters glycolysis, the TCA cycle, the pentose phosphate pathway (PPP) and glutamine metabolism in B-cell lymphoma cell lines. This analysis has revealed previously unknown pharmacological activities for rapamycin. DLCL2 (germinal-center type of diffuse large B-cell lymphoma) and Ramos (Burkitt’s lymphoma resembling activated ABC type of B-cells) cells were incubated in glucose and glutamine free DMEM containing 10% fetal bovine serum, 5 mM unlabeled or 13C-labelled glucose, 2 mM unlabeled or [U-13C5, 15N2] glutamine and rapamycin for 6 h. The steady-state labeling patterns of TCA cycle metabolites (LC-MS) and temporal profiles for labeled lactate and glutamate (NMR) were monitored. Data were analyzed with compartmentalized metabolic network models that included glucose, lactate and glutamine transport, glycolysis, TCA cycle, PPP, α-ketoglutarate-glutamate and oxaloacetate-aspartate exchange, pyruvate carboxylase activity, anaplerosis at the succinyl-CoA level, pyruvate recycling through malic enzyme, lactate dehydrogenase activity, glutaminolysis, reductive carboxylation and de novo fatty acids synthesis. Isotope balance equations were derived for every network metabolite. The resulted sets of differential equations were solved to determine flux through various metabolic pathways by isotopomer/cumomer analysis (2). Statistics for the obtained parameters/fluxes was calculated by Monte Carlo simulations including experimental noise levels. We analyzed changes in expression of ~10,000 metabolic genes in DLCL2 and Ramos cells induced by rapamycin (by RNA-seq). Anticipated related alterations indicate that mTOR inhibition diminishes glycolysis, TCA cycle activity, de novo fatty acid biosynthesis, pyruvate cycling and PPP. Glutaminolisys was also affected due to gene PPAT expression (responsible for enzymatic glutamate production from glutamine). Metabolic Flux Analysis revealed that in DLCL2 rapamycin produced dramatic metabolic changes in reductive carboxylation (41% decrease), glutaminolysis (95% decrease), mitochondrial pyruvate carrier (50% increase) and pyruvate carboxylase (520% increase) activities. Further study of these effects is required, yet these data strongly suggest that multiple pathways are affected and, potentially, these metabolic signatures may serve as key biomarkers of effective mTOR inhibition. Our genomic and proteomic analysis indicates that the expression level of a large number of enzymes in human lymphoma cells has been modified by rapamycin-mediated mTOR inhibition. These affected enzymes are involved in eight metabolic pathways: glycolysis, pyruvate cycling/metabolism, TCA cycle, glutaminolysis, fatty acid metabolism, cholesterol metabolism, phospholipid metabolism and the pentose shunt. Information available from 13C multiplets in 13C spectra and MID of numerous metabolites significantly increases precision of calculated metabolic fluxes and allows estimation of contributions of different pathways to particular metabolite labeling patterns. The new modeling approaches developed here provide more effecient methods for investigation of detailed mechanisms of cancer metabolism and therefore enable effective identification of targets for combinatorial therapy. Dynamic high-resolution MR spectra and LC-MS mass-isotopomers are very sensitive to changing/adding biochemical pathways and flux values, and metabolic modeling allows one to check precisely the feasibility of assumed general bionetworks and alterations of particular metabolic pathways.

A Kelch Domain-Containing F-Box Coding Gene Negatively Regulates Flavonoid Accumulation in Muskmelon.

The flavonoids are phenylpropanoid-derived metabolites that are ubiquitous in plants, playing many roles in growth and development. Recently, we observed that fruit rinds of yellow casaba muskmelons (Cucumis melo 'Inodorous Group') accumulate naringenin chalcone, a yellow flavonoid pigment. With RNA-sequencing analysis of bulked segregants representing the tails of a population segregating for naringenin chalcone accumulation followed by fine mapping and genetic transformation, we identified a Kelch domain-containing F-box protein coding (CmKFB) gene that, when expressed, negatively regulates naringenin chalcone accumulation. Additional metabolite analysis indicated that downstream flavonoids are accumulated together with naringenin chalcone, whereas CmKFB expression diverts the biochemical flux toward coumarins and general phenylpropanoids. These results show that CmKFB functions as a posttranscriptional regulator that diverts flavonoid metabolic flux.

Drug-Induced Rhabdomyolysis: From Systems Pharmacology Analysis to Biochemical Flux

The goal of this study was to integrate systems pharmacology and biochemical flux to delineate drug-induced rhabdomyolysis by leveraging prior knowledge and publicly accessible data. A list of 211 rhabdomyolysis-inducing drugs (RIDs) was compiled and curated from multiple sources. Extended pharmacological network analysis revealed that the intermediators directly interacting with the pharmacological targets of RIDs were significantly enriched with functions such as regulation of cell cycle, apoptosis, and ubiquitin-mediated proteolysis. A total of 78 intermediators were shown to be significantly connected to at least five RIDs, including estrogen receptor 1 (ESR1), synuclein gamma (SNCG), and janus kinase 2 (JAK2). Transcriptomic analysis of RIDs profiled in Connectivity Map on the global scale revealed that multiple pathways are perturbed by RIDs, including ErbB signaling and lipid metabolism pathways, and that carnitine palmitoyl transferase 2 (CPT2) was in the top 1 percent of the most differentially perturbed genes. CPT2 was downregulated by nine drugs that perturbed the genes significantly enriched in oxidative phosphorylation and energy-metabolism pathways. With statins as the use case, biochemical pathway analysis on the local scale implicated a role for CPT2 in statin-induced perturbation of energy homeostasis, which is in agreement with reports of statin-CPT2 interaction. Considering the complexity of human biology, an integrative multiple-approach analysis composed of a biochemical flux network, pharmacological on- and off-target networks, and transcriptomic signature is important for understanding drug safety and for providing insight into clinical gene-drug interactions.

Integrated Electroosmotic Perfusion of Tissue with Online Microfluidic Analysis to Track the Metabolism of Cystamine, Pantethine, and Coenzyme A

We have developed an approach that integrates electroosmotic perfusion of tissue with a substrate-containing solution and online microfluidic analysis of products, in this case thiols. Using this approach we have tracked the metabolism of cystamine, pantethine and CoA in the extracellular space of organotypic hippocampal slice cultures (OHSCs). Currently, little is known about coenzyme A (CoA) biodegradation and even less is known about the regulation and kinetic characteristics for this sequential multienzyme reaction. We found that the steady state percentage yields of cysteamine from cystamine and pantethine during the transit through OHSCs were 91% ± 4% (SEM) and 0.01%-0.03%, respectively. The large difference in the yields of cysteamine can be used to explain the drugs' different toxicities and clinical effectiveness against cystinosis. The kinetic parameters of the enzyme reaction catalyzed by the ectoenzyme pantetheinase are KM,C/α = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where α is the percentage yield of pantethine to pantetheine through disulfide exchange. We estimate that the percentage yield of pantethine to pantetheine through disulfide exchange is approximately 0.5%. Based on the formation rate of cysteamine in the OHSCs, we obtained the overall apparent Michaelis constant and maximum reaction rate for sequential, extracellular CoA degradation in an in situ environment, which are K'M = 16 ± 4 μM, V'max = 7.1 ± 0.5 nM/s. Kinetic parameters obtained in situ, although difficult to measure, are better representations of the biochemical flux in the living organism than those from isolated enzymes in vitro.

Workplace Health Promotion Program on Using Dietary Antioxidants (Anthocyanins) in Chemical Exposed Workers

Workplace health promotion is a business management system that aims to promote and improve the health situations of all employees in a comprehensive and sustainable mode, and should be seen as an equal partner for occupational health and safety. Certain industrial hazards (in particular chemical agents) display oxidant capacity in the human body acting via oxidative stress, caused by a cellular excess of reactive oxygen and nitrogen species. Species like superoxide and hydrogen peroxide are involved in protein kinase regulation, while nitric oxide and peroxynitrite inactivate phosphatases as well as activating kinases, which are key agents for translating signals of cell needs into metabolic change through enzyme action, gene transcription, protein synthesis, and biochemical flux. These reactions are permanently balanced by the redox system, a nonequilibrium thermodynamic state as a basic part of aerobic life, determined by relative and constantly changing amounts of oxidative and antioxidative (reductive) agents. Review of a large number of studies shows that external sources of antioxidants, mainly fruits and vegetables, are a real benefit for human defense mechanisms, considering now that sufficient scientific evidence exists for public health policy to promote a plant-rich diet. In our investigation, we focused on anthocyanins, as highly potent phytochemical antioxidants expected to protect us from oxidative injury. Furthermore, these specific antioxidants were found to act against lipoperoxidation and oxidative stress induced by industrial chemicals like carbon tetrachloride, paraquat or heavy metals. Anthocyanin extracts of Vaccinium Myrtillus (bilberry) improve night vision and eye strain caused by excessive computer use. Because of the antioxidant skin protection against UVB radiation, these phytochemicals can be used (both local and oral administration) by workers from oil-bearing platforms, furnaces or agriculture. We think that such important findings may be used for workplace health promotion projects in chemical industry, in order to prevent occupational diseases or work-related diseases. The individual approach of workplace health promotion focuses on the health-related behaviors of employees, and is called risk factor reduction (educational approach). We focused our initiative on using dietary natural antioxidants by running health campaigns, and raising awareness of the harmful effects of chemical agents and xenobiotics, addressed both to employees and managers. We propose a one year project sustaining behavior change mainly through educational and informative sessions, based on assessment of workers’ needs and also on developing healthy lifestyles. We also want to encourage company managers to offer antioxidant supplements/fresh fruits/berries juices to those employees who are working in harmful conditions, considering a daily estimated intake of 200 mg anthocyanins. Health promotion on consuming anthocyanins-rich fruits and vegetables may contribute in reducing age-related neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, age-related metabolic diseases (especially cardiovascular diseases), and not at least, cancer. Designing potent antioxidant phytococktails and standardised mixtures of anthocyanins for human consumption will be a great challenge on essentially inter-disciplinary research topic involving interaction of nutritionists, physicians, chemists, pharmacists and others.

Headspace analyses of acetone: A rapid method for measuring the 2H-labeling of water

Measurements of a 2H-labeling of water in biological fluids are required for determining the rates of biochemical flux and for estimating body composition. We have been using the method which relies on the base-catalyzed exchange of hydrogen (deuterium) between water and acetone. 2H-labeling of acetone is then determined using GCMS. Although not noted in the original paper, when chloroform is used to extract the acetone there is slow but substantial backexchange between [ 2H]acetone and solvent (unpublished observations). We report herein on a refinement of the assay that utilizes headspace analysis, which minimizes the number of transfers and decreases sample preparation time and instrument run time.

Measuring rates of biochemical flux in mice following a modified glucose tolerance test.

Insulin is an important regulator of glucose homeostasis, e.g. during a glucose challenge insulin stimulates glucose storage in the liver and the skeletal muscle. Since insulin acts on other processes, it is reasonable to expect that the syntheses of lipids and proteins are also stimulated. In cases of “insulin resistance” and Type 2 diabetes, impairments in insulin secretion/action lead to aberrant metabolic regulation. Understanding the extent to which specific pathways are affected by disease affect therapeutic options. We hypothesized that one could determine the rates of synthesis of (i) liver and muscle glycogen, (ii) fatty acids and cholesterol and (iii) proteins by administering 2H2O prior to a modified glucose tolerance test. Healthy C57BL/6J mice were fasted overnight prior to the study. On the morning of the study, mice were injected with 2H2O, randomized to receive an injection of saline (“fasted”) or a mixture of carbohydrate and essential amino acids (“fed”) and killed 3.5 h later. As compared to “fasted” mice, “fed” mice had an increase in the rates of syntheses of total liver glycogen, fatty acids, cholesterol and protein. Although further development and testing are needed, our approach may be useful in determining the metabolic response of carbohydrate, lipid and protein metabolism to a controlled challenge. Application of this method may help to resolve specific impairments in insulin action.

Oxygen and the upper limits to animal design and performance.

Mass-specific rates of aerobic metabolism VO2/Mb) scale in inverse proportion to body mass (Mb). Thus, small hummingbirds display the highest VO2/Mb known among vertebrates. Among all animals, higher VO2/Mb values are known only in flying insects. The high body-mass-specific rates of metabolism seen in hummingbirds are made possible by high lung O2 diffusing capacities, cardiac outputs, ratios of capillary surface area to muscle fiber surface area, mitochondrial volume densities, cristae surface densities and concentrations of enzymes involved in energy metabolism. Current evidence from control analyses of O2 transport through the respiratory and cardiovascular systems and of metabolic fluxes through pathways of energy metabolism indicates shared control of maximum flux rates among multiple steps (i.e. the absence of single rate-limiting steps). This supports the suggestion that functional capacities at each step in linear pathways or processes are matched to each other, and provides an explanation for why the up-regulation of functional capacities has occurred at virtually all steps in the evolution of the smallest vertebrate homeotherms. Flying insects make use of a tracheal system for O2 transport and, like hummingbirds, possess a highly up-regulated biochemical machinery for substrate oxidation. Studies of hummingbirds and honeybees reveal closer matches between biochemical flux capacities and maximum physiological flux rates than in animals capable of lower maximum VO2/Mb. It is proposed that the upper limits to functional capacities set the upper limit to VO2/Mb. This upper limit to aerobic metabolic rate may contribute, along with other factors, towards establishing the lower limit to vertebrate homeotherm size.

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

Role of Ocean in Global Environment

Ocean plays an important role in global environment in terms of heat transport and carbon cycle. Carbon is distributed in the ratio of 1:50 between atmosphere and ocean, respectively, and therefore even small fluctuation of carbon budget in the ocean will strongly affect the carbon budget in the atmosphere. Biogeochemical cycle in ocean is driven by marine ecosystem, in particular by marine phytoplankton, and complex multi-phase flow exists in the ocean. Controlled experimental ecosystem is useful in order to understand biochemical flux in multi-phase flow of marine ecosystem. Experimental results using marine phytoplankton. Emiliania huxleyi showed the importance of phytoplankton on carbon flux in the ocean.

Effect of Age and Growth Rate on Myocardial Irritability in Broiler Chickens

Young broiler chickens are undergoing a period of extremely rapid growth and may be expected to be in a state of extreme endocrine and biochemical flux. These birds are also subject to a sudden death syndrome of unknown etiology. We hypothesized that an increased myocardial sensitivity in birds exhibiting early rapid growth may contribute to this syndrome. The objective of the current study was to investigate the interaction between early growth rate and age on myocardial irritability in young broiler chickens. This study utilized 74 male broiler chickens between 3 (Group A, 21-24 days) and 6 (Group B, 43-47 days) weeks of age exhibiting rapid (heavy weight, greater than 425 g at 2 weeks) and slow (low weight, less than 350 g at 2 weeks) early growth. Physiologic parameters such as blood pressure, heart rate, and arterial blood gases in the awake, restrained birds were essentially unchanged across these groups. Myocardial irritability of the anesthetized bird (pentobarbital) as measured by the threshold to electrical fibrillation was significantly increased only in the Group A HW birds. The results of this study suggest an increased myocardial irritability in large young broilers (3 weeks of age) that is no longer present in a similar group of older birds (6 weeks of age). These findings are consistent with the hypothesis that there is an increased myocardial sensitivity in birds succumbing to sudden death syndrome, with death due to myocardial fibrillation. Further research is needed to determine the mechanisms involve.