HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

What Do We Know About Metal Recycling Rates?

The recycling of metals is widely viewed as a fruitful sustainability strategy, but little information is available on the degree to which recycling is actually taking place. This article provides an overview on the current knowledge of recycling rates for 60 metals. We propose various recycling metrics, discuss relevant aspects of recycling processes, and present current estimates on global end‐of‐life recycling rates (EOL‐RR; i.e., the percentage of a metal in discards that is actually recycled), recycled content (RC), and old scrap ratios (OSRs; i.e., the share of old scrap in the total scrap flow). Because of increases in metal use over time and long metal in‐use lifetimes, many RC values are low and will remain so for the foreseeable future. Because of relatively low efficiencies in the collection and processing of most discarded products, inherent limitations in recycling processes, and the fact that primary material is often relatively abundant and low‐cost (which thereby keeps down the price of scrap), many EOL‐RRs are very low: Only for 18 metals (silver, aluminum, gold, cobalt, chromium, copper, iron, manganese, niobium, nickel, lead, palladium, platinum, rhenium, rhodium, tin, titanium, and zinc) is the EOL‐RR above 50% at present. Only for niobium, lead, and ruthenium is the RC above 50%, although 16 metals are in the 25% to 50% range. Thirteen metals have an OSR greater than 50%. These estimates may be used in considerations of whether recycling efficiencies can be improved; which metric could best encourage improved effectiveness in recycling; and an improved understanding of the dependence of recycling on economics, technology, and other factors.     

Orientation and ingestion rates of larval Anopheles albimanus in response to floating particles

Response of fourth-instar larvae of Anopheles albimanus Wiedemann (Diptera: Culicidae) to food and inert particles floating at the water surface was studied. In a choice test, larvae aggregated at powdered organic materials (blood meal, liver powder alfalfa flour and wheat flour) but not at inert materials (kaolin, chalk or charcoal). Larvae responded positively to proteins as well as some carbohydrates, but not to cellulose. Retention of larvae at food sources found by random locomotion was found to be responsible for larval aggregation. Larvae ingested food particles 6–9 times faster than insert particles. The significance of Anopheline feeding behavior in the development of formulations of stomach toxins (bacterial agents) used in larval control is discussed.

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Zusammenfassung Die vorliegende Studie befasst sich mit Verhaltensreaktionen von Anopheles albimanus Viertlarven auf an der Wasseroberfläche schwimmende Partikel. Verteilung und Orientierung der Larven wurde in einer Wahlapparatur quantifiziert. Nach Auftrag von Alfalfamehl, Weizenmehl, Stärke, Blutmehl, Leberpulver und Fischmehl wurde Aggregation der Larven in den beköderten Fächern der Apparatur beobachtet. Sowohl Proteine (Casein) als auch einige Kohlehydrate (Amylose, Amylopectin) lösten Aggregationen der Larven aus. Im Unterschied dazu führte Auftrag von Kreide, Kaolin, Polyaethylenpulver, Talcum oder Cellulose nicht zu Aggregationen. Zur Beschreibung der Entstehung larvaler Aggregationen bei Futterstoffen wurden die Schwimmbewegungen der Larven in Anwesenheit von Weizenmehl als Ködersubstanz quantifiziert. Da keine Attraktion der Larven im Sinne einer gerichteten Schwimmbewegung beobachtet werden konnte, wird geschlussfolgert, dass sofortige Beendung der Suchaktivität der Larven bei zufällig gefundenen Futterquellen für die beobachteten Aggregationen bei organischen Substanzen verantwortlich ist.Die Fressraten der Larven bei Angebot verschiedener Substanzen im Überschuss wurde bestimmt. Larven fülten drei von insgesamt sechs Darmabschnitten innerhalb von 15–30 min bei Angebot von Futtersubstanzen, während die Füllung von nur zwei Darmabschnitten mit inerten Materialien erst nach 90–120 min zu beobachten war. Die Resultate werden in Bezug auf wasseroberflächengebundene Formulierungen von Frassgiften diskutiert. Inerte Trägersubstanzen werden wahrscheinlich wesentlich langsamer aufgenommen als Futtersubstanzen. Da An. albimanus Larven nicht von Futterquellen angezogen werden, ist eine rasche und wirksame Giftaufnahme besonders dann zu erwarten, wenn die gesamte Oberfläche der Brutgewässer mit toxinhaltigen Trägerpartikeln bedeckt werden kann.

     

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M₄₁₂) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M₄₁₂ was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M₄₁₂ which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M₄₁₂ was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M₄₁₂ without affecting the level of M₄₁₂ which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca²⁺ and their antagonists La³⁺ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.     

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

Breeding-site productivity of the swamp forest mosquitoes Mansonia bonneae and Ma.dives in Sarawak, East Malaysia

Studies on larval population densities and adult emergence rates of the Brugian filariasis vectors Mansonia bonneae Edwards and Ma. dives Schiner were conducted in freshwater swamp forest bordering the Sadong River, Serian District, Sarawak, East Malaysia, during 1984-85. Three species of aquatic host-plants in the Family Araceae were identified as supporting immature stages of the Ma. bonneae/dives complex. Proportions of positive plants were 4.7%, 6.5% and 3.4% with 6.4 +/- 2.6, 7.3 +/- 2.8 and 10.1 +/- 1.1 larvae per positive plant, respectively, for the plant species Homalomena cordata Schott, H. rostrata Griffiths and Hydrostemma motleyi (Hook. f.) Mabberley. These data indicate no significant preferences between the three types of host-plant. Detailed monitoring of the host-plant H. cordata revealed no significant monthly fluctuations in larval density per plant nor the proportion of positive plants. 11.6% of larvae were Ma. dives and 88.4% were Ma. bonneae. Mean daily yields of Ma. bonneae/dives adults per square metre of H. cordata vegetated water surface were 0.45 males plus 0.57 females during the wet season (December-February) compared with 0.2 males plus 0.31 females during the dry season (June-August). Thus output of adults per plant was approximately halved, and suitable breeding areas were further reduced, during the dry season. By extrapolation from these rates, a crude mean estimate for productivity of Ma. bonneae/dives females is 1.6 million per hectare per annum in swamp forest habitats vegetated with any of the host-plants studied.     

ELISA absorbance cut-off method affects malaria sporozoite rate determination in wild Afrotropical Anopheles

ABSTRACT. Malaria sporozoite infection rates in a mixed species group of 244 Anopheles gambiae Giles sensu lato and 115 An. funestus Giles wild female mosquitoes were compared using three methods to determine cutoff absorbance values for positivity of a Plasmodium falciparum Welch enzyme-linked immunosorbent assay (ELISA). Positive controls were based on P. falciparum circumsporozoite protein. As negative controls, four wild male Anopheles were included on each microtitre plate; tests were repeated on four consecutive days for each plate.
Infection rates were estimated at 13.1–22.8% using the mean absorbance value of negative controls plus three standard deviations, 11.7–12.8% using double the mean and 12.5–13.6% using the fixed cut-off value of 0.20 (allowing for 20% variation in negative control absorbance values).
Observed agreement for positivity or negativity among samples tested four times was 98.6% for the 2× mean method, 97.2% for the fixed cut-off 0.20 value, but only 82.7% for the mean +3 SD method. It was concluded that the 2× mean cut-off method is most reliable for field studies. P. falciparum sporozoite rates of 12.2% in An. funestus and 11.9% in An. gambiae s. l . were thus determined on the basis of the 2× mean cut-off method.
This comparative evaluation demonstrates that vector infectivity rates can be seriously over-estimated from sporozoite ELISA tests, by as much as 87% in one case considered here, depending on the absorbance cut-off method applied for negative controls.     

The Effect of Leaf Age on Gas Exchange and Malate Accumulation in C3-CAM Plant Marrubium Frivaldszkyanum (Lamiaceae)

For the first time the expression of C₃ and CAM in the leaves of different age of Marrubium frivaldszkyanum Boiss, is reported. With increasing leaf age a typical C₃ photosynthesis pattern and high transpiration rate were found. In older leaves a shift to CAM occurred and the 24-h transpiration water loss decreased. A correlation was established between leaf area and accumulation of malate. Water loss at early stages of leaf expansion may be connected with the shift to CAM and the water economy of the whole plant.