On the proposed energy switch in photosynthesis

This paper analyzes the “energy switch” that has often been proposed to direct quanta absorbed by a given photosynthetic unit alternately to the site of one and then the other primary reaction. Such a device is essential to the Franck-Rosenberg theory, but not to the Duysens-Witt-Kok (DWK) model, which needs to assume only that the reactions occur in series. If there is no energy switch, an incident quantum absorbed at any time by any particular pigment molecule stands a chance of ending up in the reactive site of either primary reaction. The “separate packages” model is a special case of this general picture. Without an energy switch, a series model requires a storage device to insure that a quantum will not be wasted if it arrives at the site of one reaction while the photosynthetic unit is set up to perform the other. Such a storage device can be appended to the DWK model. Alternatively, this model can be augmented by an energy switch. This gives what is commonly known as the “spillover model,” a confusing name which we suggest be abandoned. As a clear-cut-though perhaps technically unfeasible-test of the energy switch hypothesis, we imagine a quantum injector, a hypothetical source of flashing light which delivers a single quantum to every photosynthetic unit with each flash. We aim this useful figment at an (equally hypothetical) photosynthetic system all of whose units are set up to perform the same primary reaction. If there is an energy switch, we can now prepare a “synchronous” photosynthetic apparatus in which each photosynthetic unit is undergoing the same reaction at the same time.     

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.     

Malthusian Parameters as Estimators of the Fitness of Microbes: A Cautionary Tale about the Low Side of High Throughput

The maximum exponential growth rate, the Malthusian parameter (MP), is commonly used as a measure of fitness in experimental studies of adaptive evolution and of the effects of antibiotic resistance and other genes on the fitness of planktonic microbes. Thanks to automated, multi-well optical density plate readers and computers, with little hands-on effort investigators can readily obtain hundreds of estimates of MPs in less than a day. Here we compare estimates of the relative fitness of antibiotic susceptible and resistant strains of E. coli, Pseudomonas aeruginosa and Staphylococcus aureus based on MP data obtained with automated multi-well plate readers with the results from pairwise competition experiments. This leads us to question the reliability of estimates of MP obtained with these high throughput devices and the utility of these estimates of the maximum growth rates to detect fitness differences.     

Modifying L-type calcium current kinetics: consequences for cardiac excitation and arrhythmia dynamics

The L-type Ca current (I_Ca,L), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I_Ca,L can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I_Ca,L, modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM₁₂₃₄) in rabbit ventricular myocytes to inhibit Ca-dependent I_Ca,L inactivation, combined with the ATP-sensitive K current agonist pinacidil or I_Ca,L blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I_Ca,L blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca_i transient alternans but maintained a normal Ca_i transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca_i transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I_Ca,L can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.     

Dynamic phase microscopy reveals periodic oscillations of endoplasmic reticulum during network formation

Dynamic phase microscopy was used to study the dynamic events of formation of the endoplasmic reticulum (ER) in interphase-arrested Xenopus egg extract. We have shown that the ER periodically oscillated in an ATP-dependent manner in the frequency range of 1.6–2.2 Hz, while the tubular membrane network formed in vitro. The spectral density, i.e. the pattern of a given frequency component in the Fourier spectrum, was strongly correlated with the dynamic events during microtubule-dependent and microtubule-independent ER network formation observed by video-enhanced contrast differential interference contrast and fluorescence microscopy. Because the 1.6–2.2 Hz frequency of oscillation during the network formation was detected both in the presence and absence of microtubules, it appears to be an intrinsic ATP-dependent ER membrane property. Several characteristic active and inactive stages of ER network formation were observed both in the presence and absence of microtubules. However, data analysis of these stages indicated that microtubules and dynein motor activity have a strong influence and a cooperative effect on the kinetics of ER formation by controlled fusion reaction.     

A physical map including a new class I gene (cda12) of the human major histocompatibility complex (A2/B13 haplotype) derived from a monosomy 6 mutant cell line

To avoid interpretative problems due to restriction fragment length polymorphisms, the monosomy 6 mutant cell line BM19.7 was employed to establish a molecular map of the human major histocompatibility (HLA) complex in the A2,B13,Bw4,DRw6,DRw52,DQw1,DPw2 haplotype. Results were obtained mainly by field-inversion gel electrophoresis and Southern blotting techniques. The map extends to 4800 kb and includes the HLA complex with a length of 4200 kb. Five HTF islands could be positioned on the map. The class I region has a size of about 2000 kb and includes nonclassical HLA class I genes, some of which must be localized within 200 kb telomeric of HLA-A. A new class I gene, cda12, distinct from HLA-A, HLA-B, or HLA-C, has been localized within 50 kb from HLA-A. The class I region contains a gap of about 500 kb, just telomeric of HLA-C, in which further class I genes could not be detected. The class II region has a size of 1000 kb, which is separated from the class I region by about 1200 kb. The 5' end of the HLA-B gene is situated centromeric, giving an orientation opposite to that of the TNFA and TNFB loci. The estimated length of the HLA complex correlates well with its size determined cytogenetically using mutant cell lines with interstitial deletions.     

Resonance Raman spectroscopy of oxoiron(IV) porphyrin pi-cation radical and oxoiron(IV) hemes in peroxidase intermediates

The catalytic cycle intermediates of heme peroxidases, known as compounds I and II, have been of long standing interest as models for intermediates of heme proteins, such as the terminal oxidases and cytochrome P450 enzymes, and for non-heme iron enzymes as well. Reports of resonance Raman signals for compound I intermediates of the oxo-iron(IV) porphyrin pi-cation radical type have been sometimes contradictory due to complications arising from photolability, causing compound I signals to appear similar to those of compound II or other forms. However, studies of synthetic systems indicated that protein based compound I intermediates of the oxoiron(IV) porphyrin pi-cation radical type should exhibit vibrational signatures that are different from the non-radical forms. The compound I intermediates of horseradish peroxidase (HRP), and chloroperoxidase (CPO) from Caldariomyces fumago do in fact exhibit unique and characteristic vibrational spectra. The nature of the putative oxoiron(IV) bond in peroxidase intermediates has been under discussion in the recent literature, with suggestions that the Fe(IV)O unit might be better described as Fe(IV)-OH. The generally low Fe(IV)O stretching frequencies observed for proteins have been difficult to mimic in synthetic ferryl porphyrins via electron donation from trans axial ligands alone. Resonance Raman studies of iron-oxygen vibrations within protein species that are sensitive to pH, deuteration, and solvent oxygen exchange, indicate that hydrogen bonding to the oxoiron(IV) group within the protein environment contributes to substantial lowering of Fe(IV)O frequencies relative to those of synthetic model compounds.     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

Inhibitory action of selenite on Escherichia coli, Proteus vulgaris, and Salmonella thompson

Weiss, K. F. (Iowa State University, Ames), J. C. Ayres, and A. A. Kraft. Inhibitory action of selenite on Escherichia coli, Proteus vulgaris, and Salmonella thompson. J. Bacteriol. 90:857-862. 1965.-The resistance of three microorganisms, Escherichia coli (ISU-41), Proteus vulgaris (ISU-37c), and Salmonella thompson (ISU-86-2), to increasing concentrations of selenite was determined. E. coli was completely inhibited by 1.25% sodium hydrogen selenite, and 0.25% sodium hydrogen selenite caused a pronounced lag. P. vulgaris survived selenite concentrations of over 3%. S. thompson was inhibited completely by 3% selenite but not by 2.5%, although there was a considerable lag and a decrease in total growth. The relationship of growth, uptake, and reduction of selenite was determined. The susceptible E. coli incorporated up to twice as much selenium as did the other two organisms during the early stages of incubation. Radioautographs of seleno analogues of sulfur-containing amino acids revealed the presence of seleno-cystine in all three organisms, and seleno-methionine in E. coli. Compounds having R(F) values corresponding to possible oxidation products of seleno-methionine were present in the hydrolysates of P. vulgaris and S. thompson. Kinetic aspects of selenite uptake, rather than the ultimate localization of selenite in the cell protein, appear to be the factors that determine the degree of resistance or of susceptibility to selenite.