Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Quantitative aspects of the feeder cell phenomenon: mechanistic implications

It has been proposed that feeder cells function by supplying lymphocytes with the amino acid cysteine (a thiol compound). The results presented here indicate that thiols are the critical element of the feeder cell phenomenon. Specifically, we noted that the rank of thiol production by four different feeder cell lines corresponds to their relative abilities to support a lymphocyte cell line, CTLL-2. In addition, increasing thiol production by the feeder cells with lipopolysaccharide increased their support of CTLL-2 cells and decreasing it with homocysteate decreased support of CTLL-2 cells. However, it was also noted that substantial (up to 79% maximal) support of CTLL-2 growth was provided by feeder cell concentrations which could not produce detectable levels of free thiols. This prompted us to propose an alternative mechanism for the feeder effect which would explain these apparently paradoxical findings.     

500-MHz 1H NMR studies of ragweed allergen Ra5

The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS)     

An in-depth look at pressure sores using monolithic silicon pressure sensors

Three-dimensional scalar pressure distributions were measured in solid tissue near bony prominences in vitro in meat and in vivo in pigs using silicon pressure sensors. Data are in accord with previous theoretical models and indicate that pressure is three to five times higher internally near a bony prominence than it is at the skin over the prominence. Pressure sores are thus thought to begin internally; by the time they are evident at the skin, the sore has worked its way completely from bone to skin. This conclusion is in accord with previous clinical data. Future measurement of local vector forces is needed to fully characterize the force distribution in vivo.     

Circannual fluctuations of the serum cortisol in the European ground squirrel, Citellus citellus L

1. Serum cortisol levels were measured throughout an annual cycle in male European ground squirrels, Citellus citellus L. 2. A circannual rhythm of these levels in euthermic animals was found: the highest being in late October, November and December, the lowest in May, early October and March. 3. The levels varied in the hibernating period as well: strongly reduced during the first part (October-December) and significantly elevated in the second one (January-March). 4. A significant elevation of the cortisol levels was marked 18-20 hr after full arousal from hibernation in October.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.