Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.     

The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.     

Is ‘nocturnal hypothermia’ a valid physiological concept in small birds?: a study on Bronze Mannikins Spermestes cucullatus

The thermoregulatory capacity and metabolic responses to light–dark cycles under various mild food-deprivation treatments were measured in Bronze Mannikins Spermestes cucullatus (10–11 g). We measured the response of minimum oxygen consumption to ambient temperature in order to determine the basal metabolic rate (BMR), thermal conductance and limits of thermoneutrality of the Mannikins. In addition, we measured oxygen consumption in response to light–dark cycles and three mild food-deprivation treatments. Bronze Mannikins have a low BMR (1.67 mlO₂/g/h) that is c. 50–60% of that predicted from phylogenetically independent allometric curves for all birds. A low BMR resulted in amplitudes of metabolism between the active and rest phases that were double those predicted allometrically from body mass. The reduced nocturnal metabolic rate did not represent torpor. Typically, Mannikins would need to reduce their metabolic rate during the rest phase to c. 17% of BMR to attain the average torpor metabolic rate of other birds. The data are, however, consistent with those of other group-living Afrotropical birds that benefit energetically from group huddling in environments in which moderate seasonality is accompanied by unpredictable climates – and thus unpredictable energy inputs in time and space. When food-deprived and placed under moderate cold stress (20 °C), Mannikins decreased their rest-phase metabolic rates to the same magnitude as several small Holarctic birds. We suggest that, in the context of the progress made to quantify and define proximate heterothermic responses in endotherms, such as torpor and hibernation, the term nocturnal hypothermia often applied to moderate nocturnal reductions in metabolic rate is vague, misleading and inappropriate.     

ANALYSIS OF STARCH CONTENT IN CHLORELLA PYRENOIDOSA (CHLOROPHYTA) BY MORPHOLOGICAL AND QUANTITATIVE TECHNIQUES

Chlorella pyrenoidosa (Chick) was grown heterotrophically in batch culture on defined medium with glucose. Morphometric analysis of cells in the exponential growth phase showed that starch accounted for 57% of the volume of the chloroplast and 36% of the total cell volume. During the stationary growth phase, the amount of starch accounted for only 36% of the chloroplast volume and 13% of the total cell volume. This represented a 36% decrease in the amount of starch/cell between the exponential and stationary phase. Determination of starch as grams/cell using quantitative techniques on cell extracts showed a comparable decrease in the amount of starch during this same transition. Based on these results, morphometric techniques provided an accurate assay of starch and have the added advantage of visualization of cellular structures not available when quantitative techniques are used.     

Protoplast induction in Chlorella pyrenoidosa

Chlorella pyrenoidosa (UTEX 1230) cells in late log phase of growth were induced to form viable protoplasts by enzymatic digestion only when incubated in 2-deoxy-d-glucose (2DG) for 24 h. The combination of hemicellulase (4% w/v), Cellulysin (4% w/v), and glucuronidase (5% v/v) with 0.8 M mannitol and 8 mM CaCl₂ in modified Bristol's solution, was most effective for obtaining viable protoplasts as determined by light and electron microscopy, and vital staining with primuline (0.01% w/v). Resistance of cell walls to extensive extraction (acetolysis), and infrared analysis indicated that sporopollenin is a component of the cell wall. Transmission electron miscroscopy of acetolysed cell walls also allowed visualization of the laminate nature of the wall. This is the first report of successful induction of protoplasts from algae which contain sporopollenin in their cell walls.     

Investigation of the heterogeneity of xyloglucans from the cell walls of apple

Cell-wall material from parenchymatous tissues of apple was sequentially extracted with 50mm NaOH at 1°, m KOH at 1° and 20°, and 4m KOH at 20°, to leave a residue of α-cellulose. From the 4m KOH-soluble fraction, a crude xyloglucan was isolated by anion-exchange chromatography, and further resolved into seven xyloglucans by borate anion-exchange chromatography. The relative amounts of the xyloglucans, in order of elution, were 2.7:1.3:29.7:1.0:3.2:1.2:10.3. The structural features of five of the xyloglucans were determined by methylation analysis. These results show that apple xyloglucans exhibit heterogeneity.     

Flavonoids from the external leaf resins of four hemizonia species (asteraceae)

The external leaf resins of four Hemizonia species afforded nine methylated flavonoids, including flavanones, flavones and flavonols. Besides the rare compounds 7-methyleriodictyol and 3,6,8-trimethoxy-5,7,3′,4′-tetrahydroxyflavone the new 6-methoxy-5,7,8,3′,4′-pentahydroxyflavone was isolated and identified by spectroscopic means.     

Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects.

The exhaled breath condensate (EBC) method represents a new, noninvasive way to detect inflammatory and metabolic markers in the fluid that covers the airways [epithelial lining fluid (ELF)]. However, respiratory droplets represent only a very small and variable fraction of the EBC, most (approximately 99.99%) of which is water vapor. Our objective was to show that ELF concentrations could be calculated from EBC values by using any of three dilutional indicators (urea, total cations, and conductivity) in nine normal and nine chronic obstructive lung disease (COPD) subjects. EBC concentrations of Na(+), K(+), Ca(2+), Mg(2+), total cations, urea, and conductivity varied over a 10-fold range among individuals, but concentrations of these constituents (except Ca(2+)) remained well correlated (r(2) = 0.44-0.83, P < 0.001). Dilution (D) of respiratory droplets in water vapor was calculated by dividing plasma concentrations of the dilutional indicators by EBC concentrations. Estimates of D were not significantly different among these indicators, and urea D averaged 10,800 +/- 2,100 (SE) in normal and 12,600 +/- 3,300 in COPD subjects. Although calculated Na(+) concentrations in the ELF were less than one-half those in plasma, and concentrations of K(+), Ca(2+), and Mg(2+) exceeded those in plasma, total cation concentrations in ELF were not significantly different from those in plasma, indicating that ELF is isotonic in both normal and COPD subjects. EBC amylase concentrations (measured with an ultrasensitive procedure) indicated that saliva represented <10% of the respiratory (ELF) droplets in all but three samples. Dilutional and salivary markers are essential for interpretation of EBC studies.