On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Linkage of Pgm-3 in the house mouse and homologies of three phosphoglucomutase loci in mouse and man

The discovery of a third phosphoglucomutase locus (Pgm-3) in the house mouse is reported. Three alleles are recognized on the basis of differences in electrophoretic mobility and enzymatic activity. Pgm-3 ^a (fast mobility and high activity) is present in inbred strain C57BL/10J and 24 other strains; Pgm-3 ^b (slow mobility and high activity) is present in LP/Pas and six other strains; and Pgm-3 ^c (no detectable activity in any tissue tested) is present in strain DBA/2J and 14 other strains. Seventy-four recombinant inbred strains derived from progenitors that differed at Pgm-3 were used to study genic linkage. Pgm-3 is on chromosome 9 and is linked to Sep-1, d, Mod-1, and Ltw-3. Gene order and recombination frequencies are estimated as d 3.8±1.8% Pgm-3 2.3±1.2% Mod-1. Substrate specificities and cofactor requirements show that mouse Pgm-1 is homologous with human Pgm-2, mouse Pgm-2 with human Pgm-1, and mouse Pgm-3 with human Pgm-3.This research was supported in part by NIH Research Grant GM18684 from the National Institute of General Medical Sciences to B.A.T. and by grants from NIH A105531-02 and the Volkswagon Foundation to Jan Klein. J.H.N. was a recipient of a Fellowship from the Max-Planck-Gesellschaft, Munich. G.S. and J.K. were supported by funds from the Deutsche Forschungsgemeinschaft.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

Regulation of proton leakage from broken chloroplasts by CF0.

Measurements of proton translocation in CF₁-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF₀ is still dependent on light intensity with a first-order rate constant equal to mR₀, where R₀ is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF₀ is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF₁.CF₀ complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.     

Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggest that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.     

Studies on the binary and ternary complexes formed by a neurospora glutamate dehydrogenase and its substrates

The NADP⁺ specific glutamate dehydrogenase from wild-type $\text{Neurospora crassa}$ forms a stable binary complex with NADPH. This can combine with L-glutamate, α-ketoglutarate or the substrate analogue D-glutamate to form ternary complexes which can be distinguished by their different fluorescence properties. The affinity of the enzyme for NADPH diminishes with increases in pH or ionic strength of the solution. Experimental data obtained using modified glutamate dehydrogenases from mutant strains of $\text{N. crassa}$ suggest that the reduced-coenzyme binding sites observed fluorimetrically are the same as those observed by enzyme kinetics.     

Guidelines for model adaptation: A study of the transferability of a general seagrass ecosystem Dynamic Bayesian Networks model

In general, it is not feasible to collect enough empirical data to capture the entire range of processes that define a complex system, either intrinsically or when viewing the system from a different geographical or temporal perspective. In this context, an alternative approach is to consider model transferability, which is the act of translating a model built for one environment to another less well‐known situation. Model transferability and adaptability may be extremely beneficial—approaches that aid in the reuse and adaption of models, particularly for sites with limited data, would benefit from widespread model uptake. Besides the reduced effort required to develop a model, data collection can be simplified when transferring a model to a different application context. The research presented in this paper focused on a case study to identify and implement guidelines for model adaptation. Our study adapted a general Dynamic Bayesian Networks (DBN) of a seagrass ecosystem to a new location where nodes were similar, but the conditional probability tables varied. We focused on two species of seagrass (Zostera noltei and Zostera marina) located in Arcachon Bay, France. Expert knowledge was used to complement peer‐reviewed literature to identify which components needed adjustment including parameterization and quantification of the model and desired outcomes. We adopted both linguistic labels and scenario‐based elicitation to elicit from experts the conditional probabilities used to quantify the DBN. Following the proposed guidelines, the model structure of the general DBN was retained, but the conditional probability tables were adapted for nodes that characterized the growth dynamics in Zostera spp. population located in Arcachon Bay, as well as the seasonal variation on their reproduction. Particular attention was paid to the light variable as it is a crucial driver of growth and physiology for seagrasses. Our guidelines provide a way to adapt a general DBN to specific ecosystems to maximize model reuse and minimize re‐development effort. Especially important from a transferability perspective are guidelines for ecosystems with limited data, and how simulation and prior predictive approaches can be used in these contexts.     

Cryo-EM structure of the fatty acid reductase LuxC–LuxE complex provides insights into bacterial bioluminescence

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.     

The Nup2 meiotic-autonomous region relieves inhibition of Nup60 to promote progression of meiosis and sporulation in Saccharomyces cerevisiae

During meiosis, chromosomes undergo dramatic changes in structural organization, nuclear positioning, and motion. Although the nuclear pore complex has been shown to affect genome organization and function in vegetative cells, its role in meiotic chromosome dynamics has remained largely unexplored. Recent work in the budding yeast Saccharomyces cerevisiae demonstrated that the mobile nucleoporin Nup2 is required for normal progression through meiosis I prophase and sporulation in strains where telomere-led chromosome movement has been compromised. The meiotic-autonomous region, a short fragment of Nup2 responsible for its role in meiosis, was shown to localize to the nuclear envelope via Nup60 and to bind to meiotic chromosomes. To understand the relative contribution these 2 activities have on meiotic-autonomous region function, we first carried out a screen for meiotic-autonomous region mutants defective in sporulation and found that all the mutations disrupt interaction with both Nup60 and meiotic chromosomes. Moreover, nup60 mutants phenocopy nup2 mutants, exhibiting similar nuclear division kinetics, sporulation efficiencies, and genetic interactions with mutations that affect the telomere bouquet. Although full-length Nup60 requires Nup2 for function, removal of Nup60’s C-terminus allows Nup60 to bind meiotic chromosomes and promotes sporulation without Nup2. In contrast, binding of the meiotic-autonomous region to meiotic chromosomes is completely dependent on Nup60. Our findings uncover an inhibitory function for the Nup60 C-terminus and suggest that Nup60 mediates recruitment of meiotic chromosomes to the nuclear envelope, while Nup2 plays a secondary role counteracting the inhibitory function in Nup60’s C-terminus.