BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.     

Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA.

All retroviruses contain, in the nucleocapsid domain of the Gag protein, one or two copies of the sequence Cys-X2-Cys-X4-His-X4-Cys. We have generated a series of mutants in the two copies of this motif present in human immunodeficiency virus type 1. These mutants encoded virus particles that were apparently composed of the normal complement of viral proteins but contained only 2 to 20% of the normal level of genomic RNA. No infectivity could be detected in the mutant particles, while 10(5) infectious U were present in an equivalent amount of wild-type particles. Thus, the mutants have another defect in addition to the inefficiency with which they encapsidate genomic RNA. Our results show that both copies of the motif are required for normal RNA packaging and for infectivity. Mutants of this type may have important applications, including nonhazardous materials for research, immunogens in vaccine and immunotherapy studies, and diagnostic reagents.     

Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

Exercise effect on weight and body fat in men and women

Objectives: The effect of national exercise recommendations on adiposity is unknown and may differ by sex. We examined long‐term effects of aerobic exercise on adiposity in women and men. Research Methods and Procedures: This was a 12‐month randomized, controlled clinical trial testing exercise effect on weight and body composition in men (N = 102) and women (N = 100). Sedentary/unfit persons, 40 to 75 years old, were recruited through physician practices and media. The intervention was facility‐ and home‐based moderate‐to‐vigorous intensity aerobic activity, 60 min/d, 6 days/wk vs. controls (no intervention). Results: Exercisers exercised a mean 370 min/wk (men) and 295 min/wk (women), and seven dropped the intervention. Exercisers lost weight (women, ?1.4 vs. +0.7 kg in controls, p = 0.008; men, ?1.8 vs. ?0.1 kg in controls, p = 0.03), BMI (women, ?0.6 vs. +0.3 kg/m² in controls, p = 0.006; men, ?0.5 kg/m² vs. no change in controls, p = 0.03), waist circumference (women, ?1.4 vs. +2.2 cm in controls, p < 0.001; men, ?3.3 vs. ?0.4 cm in controls, p = 0.003), and total fat mass (women, ?1.9 vs. +0.2 kg in controls, p = 0.001; men, ?3.0 vs. +0.2 kg in controls, p < 0.001). Exercisers with greater increases in pedometer‐measured steps per day had greater decreases in weight, BMI, body fat, and intra‐abdominal fat (all p trend < 0.05 in both men and women). Similar trends were observed for increased minutes per day of exercise and for increases in maximal oxygen consumption. Discussion: These data support the U.S. Department of Agriculture and Institute of Medicine guidelines of 60 min/d of moderate‐to‐vigorous physical activity.     

Scaling-up the delivery of dog vaccination campaigns against rabies in Tanzania

An increasing number of countries are committing to meet the global target to eliminate human deaths from dog-mediated rabies by 2030. Mass dog vaccination is central to this strategy. To interrupt rabies transmission from dogs to humans, the World Health Organization recommends that vaccination campaigns should be carried out every year in all dog-owning communities vaccinating 70% of their susceptible dogs. Monitoring and evaluation of dog vaccination campaigns are needed to measure progress towards elimination. In this study, we measured the delivery performance of large-scale vaccination campaigns implemented in 25 districts in south-east Tanzania from 2010 until 2017. We used regression modelling to infer the factors associated with, and potentially influencing the successful delivery of vaccination campaigns. During 2010–2017, five rounds of vaccination campaigns were carried out, vaccinating in total 349,513 dogs in 2,066 administrative vaccination units (rural villages or urban wards). Progressively more dogs were vaccinated over the successive campaigns. The campaigns did not reach all vaccination units each year, with only 16–28% of districts achieving 100% campaign completeness (where all units were vaccinated). During 2013–2017 when vaccination coverage was monitored, approximately 20% of vaccination units achieved the recommended 70% coverage, with average coverage around 50%. Campaigns were also not completed at annual intervals, with the longest interval between campaigns being 27 months. Our analysis revealed that districts with higher budgets generally achieved higher completeness, with a twofold difference in district budget increasing the odds of a vaccination unit being reached by a campaign by slightly more than twofold (OR: 2.29; 95% CI: 1.69–3.09). However, higher budgets did not necessarily result in higher coverage within vaccination units that were reached. We recommend national programs regularly monitor and evaluate the performance of their vaccination campaigns, so as to identify factors hindering their effective delivery and to guide remedial action.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.