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Background and Objectives:Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder with a well-characterized disease phenotype but considerable interindividual heterogeneity that is not well understood. The aim of the study was to evaluate the effects of dystrophin mutations and genetic modifiers of DMD on rate and age of muscle replacement by fat.Methods:175 corticosteroid treated participants from the ImagingDMD natural history study underwent repeated magnetic resonance spectroscopy (MRS) of the vastus lateralis (VL) and soleus (SOL) to determine muscle fat fraction. MRS was performed annually in the majority of instances; however, some individuals had additional visits at 3 or 6 month intervals. Fat fraction changes over time were modeled using nonlinear mixed effects to estimate disease trajectories based on the age that the VL or SOL reached half-maximum change in fat fraction (mu) and the time required for fat fraction change (sigma). Computed mu and sigma values were evaluated for dystrophin mutations that have demonstrated the ability to lead to a mild phenotype as well as compared between different genetic polymorphism groups.Results:Participants with dystrophin gene deletions amenable to exon 8 skipping (n=4) had minimal increases in SOL fat fraction and had an increase in VL mu value by 4.4 years compared to a reference cohort (p=0.039). Participants with nonsense mutations within exons that may produce milder phenotypes (n=11) also had minimal increases in SOL and VL fat fractions. No differences in estimated fat fraction trajectories were seen for individuals amenable to exon 44 skipping (n=10). Modeling of the SPP1, LTBP4, and THBS1 genetic modifiers did not result in significant differences in muscle fat fraction trajectories between genotype groups (p>0.05); however, trends were noted for the polymorphisms associated with long-range regulation of LTBP4 and THBS1 that deserve further follow-up.Discussion:The results of this study link the historically mild phenotypes seen in individuals amenable to exon 8 skipping and with certain nonsense mutations with alterations in trajectories of lower extremity muscle replacement by fat.

Introduction Serial muscle biopsies within clinical trials for Duchenne muscular dystrophy (DMD) are critical to document therapeutic responses. Less invasive means of sampling muscle are needed. Methods We analyzed a retrospective consecutive case-series cohort of vacuum-assisted core needle muscle biopsy procedures performed on healthy and dystrophic individuals at a single institution assessing for safety and reliability of obtaining sufficient high quality biopsy tissue for histologic assessment in adult and pediatric subjects. Results Of 471 muscle cores from 128 biopsy procedures, 377-550mg of total muscle tissue was obtained per procedure with mean core weight of 129mg (SD 25.1mg). All Biopsies were adequate for histological assessment. There were no significant adverse events. Discussion This core needle biopsy approach, when combined with improved sample processing, provides a safe means to consistently obtain muscle samples for diagnostic and clinical trial applications. This article is protected by copyright. All rights reserved.

We investigate the influence of partisanship on congressional communication by analyzing 180,000 press releases issued by members of Congress (MCs) between 2005 and 2019. Specifically, we examine whether partisan factors such as party control of the White House and/or Congress influence the tone used by MCs and whether MCs are more likely to focus on issues that their respective party owns. Our analyses include the use of multiple OLS models, the machine learning approach gradient boosting, and Grimmer’s topical modeling software “expAgenda.” We find that (1) partisanship influences the tone MCs use when communicating online; and (2) MCs are unable to prioritize discussing issues that their respective party own but devote slightly greater attention to their party’s issues than MCs from the opposite party. Our study ultimately finds strong evidence of partisan influence in the way MCs design their press releases and has important implications for online congressional communication.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines. Keywords: Duchenne muscular dystrophy, combination therapy, dantrolene, dystrophin, exon skipping, armgo, ARM210, muscle, therapy

PURPOSE: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications. METHODS: From 234 subjects referred to the Undiagnosed Diseases Network, University of California–Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide. RESULTS: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality. CONCLUSION: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.

INTRODUCTION In this study we investigate associations between genotypic and sociodemographic factors and the age of diagnosis of Duchenne muscular dystrophy (DMD). METHODS Data were collected from the Duchenne Registry from 2007 to 2019, and then used to assess the impact genotype, race/ethnicity, neighborhood poverty levels, and other sociodemographics factors have on the age of diagnosis of DMD patients without a known family history, using univariate and multivariable linear regression. RESULTS The mean age of diagnosis was 4.43 years. Non-Caucasian patients and patients from high-poverty neighborhoods were older at diagnosis (P

This review aims to describe the benefits and limitations of using the Duchenne Connect patient registry to provide information particularly in regard to active treatment choices in Duchenne muscular dystrophy and their impact on disease progression.Clinical trials and natural history studies are difficult for rare diseases like Duchenne muscular dystrophy. Using an online patient self-report survey model, Duchenne Connect provides relevant data that are difficult to gather in other ways. Validation of the overall dataset is supported by comparable mutational spectrum relative to other cohorts and demonstrated beneficial effect of corticosteroid use in prolonging ambulation. These types of analyses are provocative and allow multivariate analyses across the breadth of patient and physician medication and supplement practices. Because the data are self-reported and online, the barrier to participation is low and great potential exists for novel directions of further research in a highly participatory forum.Patient registries for Duchenne and Becker muscular dystrophy (DBMD) are powerful tools for monitoring patient outcomes, comparing treatment options, and relating information between patients, researchers, and clinicians. Duchenne Connect is an online patient self-report registry for individuals with DBMD that facilitates aggregation of treatment modalities, outcomes, and genotype data and has played a vital role in furthering DBMD research, particularly in the USA, in a highly participatory and low-cost manner.

To assess the utility of online patient self-report outcomes in a rare disease, we attempted to observe the effects of corticosteroids in delaying age at fulltime wheelchair use in Duchenne muscular dystrophy (DMD) using data from 1,057 males from DuchenneConnect, an online registry. Data collected were compared to prior natural history data in regard to age at diagnosis, mutation spectrum, and age at loss of ambulation. Because registrants reported differences in steroid and other medication usage, as well as age and ambulation status, we could explore these data for correlations with age at loss of ambulation. Using multivariate analysis, current steroid usage was the most significant and largest independent predictor of improved wheelchair-free survival. Thus, these online self-report data were sufficient to retrospectively observe that current steroid use by patients with DMD is associated with a delay in loss of ambulation. Comparing commonly used steroid drugs, deflazacort prolonged ambulation longer than prednisone (median 14 years and 13 years, respectively). Further, use of Vitamin D and Coenzyme Q10, insurance status, and age at diagnosis after 4 years were also significant, but smaller, independent predictors of longer wheelchair-free survival. Nine other common supplements were also individually tested but had lower study power. This study demonstrates the utility of DuchenneConnect data to observe therapeutic differences, and highlights needs for improvement in quality and quantity of patient-report data, which may allow exploration of drug/therapeutic practice combinations impractical to study in clinical trial settings. Further, with the low barrier to participation, we anticipate substantial growth in the dataset in the coming years.

To assess the utility of online patient self-report outcomes in a rare disease, we attempted to observe the effects of corticosteroids in delaying age at fulltime wheelchair use in Duchenne muscular dystrophy (DMD) using data from 1,057 males from DuchenneConnect, an online registry. Data collected were compared to prior natural history data in regard to age at diagnosis, mutation spectrum, and age at loss of ambulation. Because registrants reported differences in steroid and other medication usage, as well as age and ambulation status, we could explore these data for correlations with age at loss of ambulation. Using multivariate analysis, current steroid usage was the most significant and largest independent predictor of improved wheelchair-free survival. Thus, these online self-report data were sufficient to retrospectively observe that current steroid use by patients with DMD is associated with a delay in loss of ambulation. Comparing commonly used steroid drugs, deflazacort prolonged ambulation longer than prednisone (median 14 years and 13 years, respectively). Further, use of Vitamin D and Coenzyme Q10, insurance status, and age at diagnosis after 4 years were also significant, but smaller, independent predictors of longer wheelchair-free survival. Nine other common supplements were also individually tested but had lower study power. This study demonstrates the utility of DuchenneConnect data to observe therapeutic differences, and highlights needs for improvement in quality and quantity of patient-report data, which may allow exploration of drug/therapeutic practice combinations impractical to study in clinical trial settings. Further, with the low barrier to participation, we anticipate substantial growth in the dataset in the coming years.

Objective: The changing effectiveness of a treatment program for dual-diagnosis patients was evaluated over a 2-year period with the use of a sequential study group design. Method: The treatment outcome of 179 consecutively enrolled patients with chronic psychotic illness and comorbid substance dependence who entered a specialized day hospital dual-diagnosis treatment program from Sept. 1, 1994, to Aug. 31, 1996, was evaluated. The 24 months were divided into four successive 6-month periods for comparing the evolving effectiveness of the program for groups of patients entering the day hospital during these four periods. Treatment attendance, hospital utilization, and twice weekly urine toxicology analyses were used as outcome measures. Results: The initial treatment engagement rate, defined as at least 2 days of attendance in the first month, increased significantly from group 1 to group 4, more than doubling. Thirty-day and 90-day treatment retention rates also substantially increased from group 1 to group 4. More patients had no hospitalization in the 6 months after entering the day hospital program than in the 6 months before entering the day hospital program. Urine toxicology monitoring indicated that the patients in group 4 were more likely than those in group 1 to remain abstinent at follow-up. Conclusions: The evolving clinical effectiveness of a developing program can be quantified by using a sequential group comparison design. The sequential outcome improvements may be related to the incremental contributions of assertive case management and skills training for relapse prevention. (Am J Psychiatry 1999; 156:1765‐1770)

Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.

Background There is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped cis and trans regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs). Results Unexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an Xist independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained trans ceQTLs with -log10 P > 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements. Conclusion Radiation hybrids are a useful tool for high resolution mapping of cis and trans loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.

Using radiation hybrid genotyping data, 99% of all possible gene pairs across the mammalian genome were tested for interactions based on co-retention frequencies higher (attraction) or lower (repulsion) than chance. Gene interaction networks constructed from six independent data sets overlapped strongly. Combining the data sets resulted in a network of more than seven million interactions, almost all attractive. This network overlapped with protein–protein interaction networks on multiple measures and also confirmed the relationship between essentiality and centrality. In contrast to other biological networks, the radiation hybrid network did not show a scale-free distribution of connectivity but was Gaussian-like, suggesting a closer approach to saturation. The radiation hybrid (RH) network constitutes a platform for understanding the systems biology of the mammalian cell.

Meiotic mapping of quantitative trait loci regulating expression (eQTLs) has allowed the construction of gene networks. However, the limited mapping resolution of these studies has meant that genotype data are largely ignored, leading to undirected networks that fail to capture regulatory hierarchies. Here we use high resolution mapping of copy number eQTLs (ceQTLs) in a mouse-hamster radiation hybrid (RH) panel to construct directed genetic networks in the mammalian cell. The RH network covering 20,145 mouse genes had significant overlap with, and similar topological structures to, existing biological networks. Upregulated edges in the RH network had significantly more overlap than downregulated. This suggests repressive relationships between genes are missed by existing approaches, perhaps because the corresponding proteins are not present in the cell at the same time and therefore unlikely to interact. Gene essentiality was positively correlated with connectivity and betweenness centrality in the RH network, strengthening the centrality-lethality principle in mammals. Consistent with their regulatory role, transcription factors had significantly more outgoing edges (regulating) than incoming (regulated) in the RH network, a feature hidden by conventional undirected networks. Directed RH genetic networks thus showed concordance with pre-existing networks while also yielding information inaccessible to current undirected approaches., Author Summary An important problem in systems biology is to map gene networks, which help identify gene functions and discover critical disease pathways. Current methods for constructing gene networks have identified a number of biologically significant functional modules. However, these networks do not reveal directionality, that is, which gene regulates which, an important aspect of gene regulation. Radiation hybrid panels are a venerable method for high resolution genetic mapping. Recently we have used radiation hybrids to map loci based on their effects on gene expression. Because these regulatory loci are finely mapped, we can identify which gene turns on another gene, that is, directionality. In this paper, we constructed directed networks from radiation hybrid expression data. We found the radiation hybrid networks concordant with available datasets but also demonstrate that they can reveal information inaccessible to existing approaches. Importantly, directionality can help dissect cause and effect in genetic networks, aiding in understanding and ultimately rational intervention.

We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse–hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The −2log10P support interval for the ceQTLs was 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome.

Eyes of glasseyes (Priacanthidae) show conspicuous eyeshine and have a brilliant tapetum in the chorioid. The tapetum underlies the entire retina; it is composed of several rows of reflecting cells which contain stacks of flat crystals lying parallel to the retinal surface in the central fundus and obliquely towards the periphery. Reflexion is orange and specular; the reflexion spectrum is a broad band centred at about 630 nm. Processes of the pigment epithelium contain black pigment in some parts of the eye; pigment is especially dense in a horizontal central band and in the lower field, but is absent from the cell bases. The ultrastructure of the pigment epithelium and of the tapetum is described. The crystals and intervening cytoplasmic lamellae are organized as quarter-wavelength films to give maximal reflexion of long wavelengths (orange and red light) by constructive interference. Rods and cones are present; there is no retinomotor activity. The mechanism of reflexion, efficiency of the tapetum and role of the retinal pigment are discussed.

Eyes of four species of gars were studied, namely, alligator (Lepisosteus spatula Lacépède), spotted (L. oculatus (Winchell)), shortnose (L. platostomus Rafinesque), and longnqse (L. osseus (L.)). The retina is duplex, a yellow tapetum lucidum is present, rods and retinal pigment move, and the tapetum is uncovered in dim light. The visual pigment is a porphyropsin, λmax 523, and the optical density of the pigment in the retina is 0.22. Transmission through the retina is low at short wavelengths and rises steeply above 460 nm. Reflectance from the tapetum is diminished in the blue, and rises progressively at longer wavelengths to reach a maximum (70%) at about 650 nm. Underwater illumination was measured in waters where gars occur. In lake waters maximal transmissivity occurred at 375 nm and the transmission per meter ranged from 76 to 62%. In rivers and bays, transmission was maximal at long wavelengths, ≥600 nm, and the transmission per meter ranged from 37% to 0.001%; these waters sometimes were stained, and were fairly turbid to very muddy. The yellow tapetum is an efficient reflector of underwater light, the greater part of the energy of which occurs at long wavelengths. It is calculated that the tapetum increases light absorption in the retina by 30%. In muddy rivers inhabited by gars, scotopic vision involving the tapetum probably operates during the daytime at depths of 2 m or less.

— From kinetic analysis of the Z-scheme we have derived expressions for fractions of open reaction centers and throughput quantum yield. The fraction of absorbed quanta available to photoreaction II has been treated by the wavelength dependent function, α. However, for purposes of analysis in terms of throughput electron flow it is necessary to introduce a related function, α', for actual processing of excitations by photoreaction II; α' differs from α if the individual quantum yields of the two photoreactions are not equal. ‘Included in our findings are the following. As a close approximation quantum yield is maximum at α’= 0.5 and a symmetrical function of α' around the line α' =0.5. As previously noted, lowering of the apparent equilibrium constant between the photoreactions flattens dependence of quantum yield on α' near α' = 0.5 but also lowers the attainable quantum yield. For any given equilibrium constant the effective fractions of open reaction centers are equal at α' = 0.5 and mirror images of each other around the line α' = 0.5. Slow changes in quantum yield following changes in wavelength (the State 1-State 2 phenomenon) are explainable in terms of changes in α' which also have the effect of flattening dependence of quantum yield on wavelength. We have used these changes to estimate the neutral wavelength for α' = 0.5 as 681–682 nm in Chlorella.

Growth of Anacystis nidulans (Richt.) Drouet & Daily in wavelengths of light predominantly absorbed by chlorophyll a causes a dramatic lowering in chlorophyll content and in the chlorophylllphycocyanin ratio. A limit to the effect is reached in far red (680 nm) light where the ratio chlorophylllphycobilinogen falls to 650 nm)filters; it gave the same extreme pigment ratio together with a high specific growth rate of 2.5 day-1. Compared with normally pigmented cells there are three features which accompany the low-chlorophyll condition of far red light. At 677 nm relative quantum efficiency increases but action decreases. Synthesis of total pigment and total cell material in far red light also decreases. These observations suggest that the low chlorophyll response to far red light reflects an incompetence rather than an adaptation.

Sperm and eggs of sand dollars, Melitta quinquiesperforata (Leske), were subjected to two petroleum oils, and effects determined. The oils chosen were Kuwait crude and No. 2 fuel oil, supplied by the American Petroleum Institute. Water-soluble extracts (WSF) from oil-sea water mixes were prepared and the major aromatic components in the WSF of the fuel oil were identified. WSF of No. 2 fuel oil depressed respiration, mobility of sperm, interfered with fertilization and cleavage, and retarded larval development. The effects were detectable at dilutions of 4% and less (about 0.6 ppm of WSF). Kuwait crude was much less toxic. There was no effect on water permeability of the egg membrane. Results are compared with similar studies on other marine organisms.

The effects of the water-soluble fractions (WSF) of petroleum oils and of solutions of aromatics on embryos and nauplii of barnacles (Chthamalus fragilis and Balanus amphitrite niveus) were investigated. The oils tested were S. Louisiana, Alaska, Kuwait, Venezuela crudes, Diesel fuel, Bunker C, No. 2 fuel and crankcase oils. Eighteen aromatic hydrocarbons occurring in petroleum oils were also tested. Observations were made on development and hatching of embryos, and the activity, phototaxis and survival of larvae. Acute experiments (1 h duration) were carried out in glass tubes illuminated above, and larvae remaining on the bottom were separated from those actively swimming. Concentrations at which half the larvae occurred in the bottom fraction were determined. Oils were toxic in the series used: crank case > No. 2 fuel oil > Bunker C > Diesel > Venezuela > Kuwait > Alaska > S. Louisiana (in terms of percentages of WSF). Relative toxicities of the aromatics (in terms of percentages of saturated solutions) are given. Embryonic development and larval activity were adversely affected by No. 2 fuel oil at a concentration of 3 ppm and larval activity by naphthalene at the same level.

We explored O 2 flash yield in two cyanophytes, Anacystis nidulans and Agmenellum quadruplicatum . On a rate-measuring electrode, a single flash gave a contour of O 2 evolution with a peak at about 10 ms which was maximum (100) for 680 nm background light. On 625 nm illumination the peak was smaller (62) but was followed by an increased tail of O 2 attributed to enhancement of the background. After a period of darkness, repetitive flashes (5 Hz) gave a highly damped initial oscillation in individual flash yields which finally reached steady state at 94% of the yield for 680 nm illumination. When O 2 of repetitive flashes was measured as an integrated flash yield the results was distinctive and similar to that for a continuous light 1 (680 nm). An apparent inhibition of respiration which persisted into the following dark period was taken as evidence for the Kok effect. With a concentration-measuring electrode, integrated flash yield vs. flash rate showed the same nonlinear behavior as O 2 rate vs. intensity of light 1. We draw three conclusions about the two cyanophytes. (a) The plastoquinone pool is substantially reduced in darkness. (b) Because of a high ratio of reaction centers, reaction center 1 / reaction center 2, for the two photoreactions, saturating flashes behave as light 1. (c) Because repetitive flashes are light 1, they also give a Kok effect which must be guarded against in measurements designed to count reaction centers.

Spontaneous pigment mutants of Anacystis nidulans were self-selected for improved growth in far red light (> 650 nanometers). Questions were asked about those features of the light-harvesting mechanism which altered to give the mutants improved photosynthetic performance in far red. Answers were sought by comparing pigment and reaction center concentrations for the parent and six mutants grown in gold fluorescent and in far red light. Three significant results emerged. The ratio of reaction centers for photoreactions I and II (RC1/RC2) varied by a value of about 2.1 for all clones grown in gold and a value of about 1.1 for all clones grown in far red. Alteration of the ratio was not evident in any of the mutants.Phycobilisome alterations were evident as decreased phycocyanin content in all mutants. In three mutants, allophycocyanin became the major remaining phycobilisome component. Action spectra for photoreactions I and II allowed estimates of chlorophylls serving each of the two reaction centers. Ratios of chlorophylls to reaction centers within each photosystem were chlorophyll I/RC1 = 118 +/- 11 and chlorophyll II/RC2 = 52 +/- 9 for all seven clones grown in both gold and far red light. Remarkable constancy of these ratios, in spite of wide variation in cell material, supports an hypothesis that in A. nidulans there are two chlorophyll proteins, each bearing a reaction center and chlorophylls in fixed ratio.

— We have devised a method of obtaining simultaneous action spectra for photoreactions I and II by analysis of direct and indirect effects involved in enhancement. The method requires previous determination of the neutral wavelength which gives maximum quantum yield by virtue of equal fractions of open reaction centers (p and q) for each photoreaction. A sufficient intensity of the neutral wavelength is used as a constant background. Upon addition of a weak modulated measuring light of intensity Im and wavelength λm two amperometric signals are obtained for rate of oxygen evolution. A modulated signal (AC¯) isolates the direct effect of Im and gives action of photoreaction II as AC/Im. An increment in total rate (ΔDC) also includes an indirect effect of Im in perturbing reaction center conditions (p and q). From analysis of interaction of the two photoreactions, action for photoreaction I can be estimated as (2 ΔDC-AC)/Im. The method is applicable to whole cells, properly scales the two action spectra to each other, and removes contribution of the State 1-State 2 phenomena. Action spectra were obtained for Chlorella.

— Action spectra for photoreactions I and II of photosynthesis were obtained for Anacystis nidulans and three of its variants which had altered chlorophyll/phycocyanin ratios. The spectra are properly scaled to each other. They provide information on contributions of phycocyanin and chlorophyll to initial absorption and final distribution of excitation energy to reaction centers I and II. In normally pigmented cells the light harvesting pigments for photoreaction I include about 40% of the phycocyanin and 84% of the chlorophyll. Both in normal cells and in cells with altered pigmentation excitation energy from phycocyanin is delivered to photoreaction II via a small number of chlorophylls. In response to alterations in chlorophyll/phycocyanin ratio Action I spectra showed large variations whereas Action II spectra were essentially invariant. The result is taken to mean that alteration in chlorophyll components in Anacystis is attended by a special restriction: there are only small changes in amount of chlorophyll accessible to photoreaction II in the face of large changes in amount committed to photoreaction I.

Eyes of cusk-eels Ophidion welshi and Lepophidium graellsi contain mostly short rods, arranged in five or six regular rows. In O. welshi, cones are single and double, whereas cones are lacking in L. graellsi. The pigment epithelium contains a lipid tapetum lucidum. In L. graellsi the tapetal spheres are 465 nm in diameter; in O. welshi most tapetal spheres have diameters of 187 nm; others, less frequent, are 86 nm. The tapetum of L. graellsi is white, that of O. welshi is blue; they reflect diffusely and reflectance at λmax is about 0.60. Increased tonicity causes the tapetum of O. welshi to turn darker blue and reduced tonicity causes it to become white. The lipid of the tapetum was examined by thin-layer and gas–liquid chromatography; it is a triglyceride containing mostly (88%) docosahexaenoic acid. The visual pigments are rhodopsins; L. graellsi λmax 494, O. welshi λmax 500 nm. Coastal waters where cusk-eels live were fairly turbid: maximal transmission of daylight occurred at 585 nm when the extinction coefficient was 0.6. Reference is made to the secretive and nocturnal habits of cusk-eels, and the functioning of their unusual eyes.

— –The method of Weber and Teale for determining absolute fluorescence quantum yield of dyes in solution was modified for determination of the yield of bacteriochlorophyll fluorescence from chromatophores and whole cells of photosynthetic bacteria. Measured yields ranged from about 1–6 per cent. The yield depended on intensity and wavelength of the exciting light. The higher yield at higher light intensity was interpreted as due to saturation of photosynthesis. The lower yield in some strains when excited at 810 nm was attributed to preferential excitation of the reaction center pigment P800. From this study and the lifetime measurements of others, the relation τ=Q.τ0 was substantiated for the fluorescence of bacteriochlorophyll in vivo, τ being the actual lifetime, τ0 the intrinsic lifetime as estimated from the absorption band area, and Q the quantum yield of fluorescence.

Summary Lauryl dimethyl amine oxide was used to isolate reaction centers from a carotenoid-less mutant, strain G-9, of Rhodospirillum rubrum. These reaction centers have absorption spectra and light or chemically induced difference spectra very similar to those obtained from Rhodopseudomonas spheroides, strain R-26. But, unlike those from Rps. spheroides, they are more labile to higher detergent concentrations and to ammonium sulfate. The cytochrome content was estimated to be less than one per 10 P870.

Emission spectra and transient behavior of fluorescence in Porphyridium cruentum have been studied in search of the pathway of excitation energy from the phycobilisome to Photosystem I (PS I) of photosynthesis. For activating light at 436 nm, absorbed almost entirely by chlorophyll, fluorescence is dominated by the 718 nm band generally attributed to chlorophyll of PS I. Activating light at 550 nm, absorbed mostly by the phycobilisome, gives rise to the distinctive fluorescence band of PS II chlorophyll at 696 nm but also gives a large component at 718 nm. Analysis depends critically upon the source of emission at 718 nm under 550 nm activation: does it arise from PS I or PS II? Ley and Butler (Ley, A.C. and Butler, W.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3956–3960) have proposed that the 718 nm arises mostly from PS I, to which it is transferred by spillover from PS II. We suggest a different proposition: that under 550 nm activation most of the 718 emission arises from PS II. Analysis shows that this proposition provides an alternative explanation. Using the small change in fluorescence yield observed under 436 nm activation as a monitor of excitation in PS I, we provide evidence that under 550 activation most of the 718 nm fluorescence arises from PS II.

Under far-red (greater than 650 nm) illumination Anacystis nidulans grows poorly and develops a low chlorophyll content. During continued culture over many generations there are increases in growth rate and in the chlorophyll/phycocyanin ratio, usually occurring in concomitant and stepwise fashion. From such selection cultures six clones have been established which differ from the parent in pigment content and show improved growth rate in far-red light. From the evidence at hand the six clones are presumed to be spontaneous mutants selected under the photosynthetically restrictive condition of far-red illumination.

Publisher Summary This chapter discusses the amperometric hydrogen electrode. It describes the use of a commercially available Clark-type electrode for measurement of hydrogen in aqueous solution. The electrode is applicable to the determination of biological hydrogen production. At a platinum electrode maintained at proper potential, hydrogen is oxidized. An apparatus consisting of OX 700 Clark-type electrode and OX 705 water-jacketed cuvette from Gilson Medical Electronics may be used electrochemical detection of hydrogen. The electrode contains a small platinum wire at the center surrounded by a much larger reference silver electrode. A simple voltage divider consisting of two properly chosen resistors can be used to obtain a fixed polarizing voltage from a mercury battery. A potentiometer should be used if a change in polarizing voltage is desired. The resistance in the divider chain should be chosen so that the current flowing through the divider chain is at least 100 times greater than the maximum anticipated electrode current. The slopes of recorded signals versus time are used to compute the rates of hydrogen evolution.

The State 1-State 2 phenomenon of photosynthesis was studied in Chlorella by measuring the flash yield (Y) and the modulated rate ( v ) of oxygen evolution induced by weak modulated 650-nm light. From light intensity curves, intensities of 650 and 710 nm background and preilluminations were chosen to give maximum values of Y and v . Following long preilluminations in 710 nm (State 1) or in 650 nm (State 2), Y and v were measured in background light of chosen wavelength. The resulting plots of v vs Y show a discontinuity between State 1 and State 2. They confirm the predictions of Bonaventura and Myers [(1969) Biochim. Biophys. Acta 189, 366–383] and are consistent with changes in α (fraction of absorbed light captured by System II) as explanation of the State 1–State 2 phenomenon. When the intensity of 710 nm preillumination was too low, the characteristics of State 1 were not fully developed and the results then were similar to those of Delrieu [(1972) Biochim. Biophys. Acta 256, 293–299].

Publisher Summary This chapter discusses the photochemical reaction centers from Rhodopseudomonas spheroids . The hallmark of the known bacterial photosynthetic reaction centers is the presence of a bacteriochlorophyll (BChl) molecule that is specialized to act as a primary photochemical electron donor. This BChl, called “P870,” “P890,” and so on, after the wavelength of its long-wave absorption maximum, is oxidized by light, while an unspecified electron acceptor becomes reduced. Oxidation of P870 is signaled by loss of the long-wave absorption band (bleaching). In living cells, the oxidized P870 is reduced by one or more c -type cytochromes. In purified reaction centers, the source of electrons for the re-reduction of oxidized P870 depends on the environment. In any case, the defining assay for reaction centers is the reversible light-induced bleaching of P870. In the reaction centers made from Rhodopseudomonas spheroides or Rhodospirillum rubrum , the bleaching of P870 is accompanied by blue shift of a band near 800 nm because of another specialized BChl, P800. There appear to be two or three P800 for each molecule of P870. The absorption increase at 780 nm caused by this blue shift is often easier to measure than the bleaching at 870 nm. Rhodopseudomonas viridis contains the longer-wave pigment BChl b . Reaction centers made from this organism contain P830 and P960, analogous to the P800 and P870 of Rhodopseudomonas spheroides . The blue shift of P830 can be measured as an increase in optical density at 810 nm.

During growth under far-red (>650 nm) light, Anacystis nidulans accumulates protochlorophyllide to concentrations about one-tenth of the chlorophyll. From whole cell fluorescence spectra, protochlorophyll(ide) was identified also in another blue-green, and in a red, alga grown in far-red light.