The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

The effect of microsomal enzyme inducing drugs on reproductive function in the ewe.

A number of drugs cause marked increases in the steroid hydroxylase activity of hepatic microsomes. Beginning 2 days after estrus, 117 mature ewes were each given 14 injections over a 27-day period of phenobarbital sodium, diphenylhydantoin, chlorcyclizine HCl or phenylbutazone. Blood samples for luteinizing hormone (LH) and progesterone determination by radioimmunoassay (RIA) were taken on day 10 of the first estrous cycle (day 18 if no heat was observed) and on days 5 and 10 of the second cycle. On day 10 of the second cycle, the ewes were given an intravenous injection of 1 ml of 6% solution of pentobarbitol sodium anesthetic per 4.5 kg body weight, and the length of anesthetic sleep time was measured. The ewes were then killed and corpora lutea and liver were weighed.In 33 ewes treated with either phenobarbitol sodium or phenylbutazone, sleep time was shortened (18 min $\text{vs}$ 29 min in untreated controls, P<.01), indicating that enzyme induction had occurred. For 41 ewes treated with either chlorcyclizine HCl or diphenylhydantoin, sleep time was lengthened to 93 min (P<.01 $\text{vs}$ controls), indicating impaired liver function. Electron micrographs of liver cells verified that enzyme induction or hepatic degeneration had occurred.     

Reconstitution of the Na+K+ pump of Ehrlich ascites tumor and enhancement of efficiency by quercetin

Plasma membranes from Ehrlich ascites tumor cells were solubilized by octylglucoside in the presence of phospholipids. The Na+K+-ATPase was purified from this extract by adsorption and elution from thio-Seph-arose 4B. The enzyme (specific activity, 7 mumoles of ATP hydrolyzed min-1 mg of protein -1) was reconstituted into liposomes by the octyglucoside dilution procedure. An ATP-dependent Na+ influx with low efficiency was observed. On addition of appropriate amounts of quercetin, the Na+ flux/ATP hydrolysis ratio was increased from 0.4 to 1.4.     

Spectral composition of shade light in coastal-zone oak forests in SE Bulgaria,and relationships with leaf area index: a first overview

Major knowledge gaps exist with respect to light-quality regimes in the coastal-zone Strandzha Quercus frainetto (Q.f.) forest region adjoining the southern Bulgarian Black Sea. This paper presents preliminary results that help narrow these gaps. In conjunction with leaf area index (LAI) field campaigns we undertook measurements with an array of 7 broad-band (ca 40 nm) sensors covering the range 0.40–0.94 μm, plus 1 sensor for UVB (0.297 μm peak) and 1 for photosynthetically active radiation (PAR). Measurements focused on inside-forest shade conditions at sites 0 to ca 15 km from the Black Sea and at altitudes up to ca 120 m above sea level. Some of the sites were also studied using a high-resolution spectroradiometer. A sequential measuring strategy was necessary. This involves potentially large uncertainties, here addressed through estimations of the variability around the sinusoidal course of daylight. Light-quality regimes were found to be in general support of earlier studies of deciduous forests. Our data from the broad-band sensors and from the spectroradiometer are mutually supportive. They indicate a stronger red-shift below Q.f. canopies than below canopies in enclaves dominated by Fagus orientalis and Pinus sylvestris. Transmission in the range 0.50–0.55 μm increases beneath the three types of canopies, most pronounced in the Q.f. case. Analysis of relationships between the inside-forest to open-field irradiance ratio and LAI supports the use of Beer’s Law. We found a fairly strong relationship between the red (0.66 μm) to far-red (0.73 μm) irradiance ratios (R/FR) and LAI for the Q.f. forest. In quantitative terms, the result is new for this Q.f. region, and suggests further research to explore whether a two-sensor approach (0.66 and 0.73 μm) might offer possibilities for further low-cost mapping of the spatio-temporal patterns of R/FR and LAI in Strandzha. Such mapping would assist in further studies of the region’s forest biogeochemistry and vitality.     

The effects of manipulation of sedimentary iron and organic matter on sediment biogeochemistry and seagrasses in a subtropical carbonate environment

The microbial metabolism of organic matter (OM) in seagrass beds can create sulfidic conditions detrimental to seagrass growth; iron (Fe) potentially has ameliorating effects through titration of the sulfides and the precipitation of iron-sulfide minerals into the sediment. In this study, the biogeochemical effects of Fe availability and its interplay with sulfur and OM on sulfide toxicity, phosphorous (P) availability, seagrass growth and community structure were tested. The availability of Fe and OM was manipulated in a 2 × 2 factorial experiment arranged in a Latin square, with four replicates per treatment. The treatments included the addition of Fe, the addition of OM, the addition of both Fe and OM as well as no addition. The experiment was conducted in an oligotrophic, iron-deficient seagrass bed. Fe had an 84.5% retention efficiency in the sediments with the concentration of Fe increasing in the seagrass leaves over the course of the experiment. Porewater chemistry was significantly altered with a dramatic decrease in sulfide levels in Fe addition plots while sulfide levels increased in the OM addition treatments. Phosphorus increased in seagrass leaves collected in the Fe addition plots. Decreased sulfide stress was evidenced by heavier δ³⁴S in leaves and rhizomes from plots to which Fe was added. The OM addition negatively affected seagrass growth but increased P availability; the reduced sulfide stress in Fe added plots resulted in elevated productivity. Fe availability may be an important determinant of the impact that OM has on seagrass vitality in carbonate sediments vegetated with seagrasses.     

Sharp intense line in the bioluminescence emission of the firefly

Numerous investigations have been carried out on the spectral distribution of the light of different species of fireflies. Here we record the emission spectrum of the Indian species of the firefly Luciola praeusta Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae) on a color film. Green and red color-sectors, with an intense yellow one in between, appear in this spectrum. Intensity profile of this spectrum reveals a hitherto undetected strong narrow yellow line, which lies within the full-width-at-half maximum (FWHM) of the intensity profile. The spectrum recorded in a high-resolution spectrometer confirms the presence of this sharp intense line. This finding lends support to an earlier drawn analogy between the in vivo emission of the firefly and laser light.