Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krüppel

We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post-translational modification of proteins.     

A new method for the isolation of fresh hepatocytes from periportal and pericentral regions of the liver lobule

A simple method which avoids the use of perfusion with calcium free buffer, hydrolytic enzymes and detergents has been developed to obtain fresh hepatocytes from periportal and pericentral regions of the liver lobule. Cylindrical plugs (200 x 500 microns) of periportal and pericentral areas of the rat liver lobule weighing about 1 mg were collected with a micropunch from fresh or perfused liver. Ninety percent of cells were intact as assessed from trypan blue staining. Glutamine synthetase activity was detected predominantly (ca. 85%) in plugs isolated from pericentral regions indicating that this method allows selective harvesting of pure sublobular zones of the liver lobule. Rates of oxygen uptake measured at 25 degrees C by plugs from livers perfused in the anterograde direction were 56 +/- 5 and 33 +/- 7 mumol/g/h by periportal and pericentral plugs, respectively, values similar to data obtained from the intact organ. This method provides new opportunities to study the regulation of basic metabolic processes in cells from sublobular areas under nearly physiological conditions.     

The influence of ion species on phosphatidylcholine bilayer structure and packing

The effects of various monovalent cations and anions on the bilayer packing and structure of dipalmitoylphosphatidylcholine were studied using X-ray diffraction and differential scanning calorimetry. It was observed from the X-ray diffraction studies that monovalent salts, in general, have no effect on bilayer packing. The results of DSC studies on metal chloride systems are consistent with the interpretation that cations in general and Li+ in particular bind to DPPC bilayers. The effect of potassium salts on pre- and main-transition temperatures suggest that anions, such as Acetate-, also significantly bind to DPPC head groups.     

Phenolic antioxidants: potent inhibitors of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

Bis(2-hydroxy-3-tert-butyl-5-methylphenyl)methane (bis-phenol) is the most potent inhibitor of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum yet identified. The compound behaves as a reversible, tight-binding inhibitor with apparent Ki = 0.3 microM. Butylated hydroxytoluene, butylated hydroxyanisole, and 4-nonylphenol are also effective inhibitors. These observations are of particular interest in light of the widespread use of such phenolic antioxidants and stabilizers in the food industry and in the manufacture of rubbers and plastics and the ease with which the compounds are extracted into organic solvents.     

Characterization of an associated equilibrium folding intermediate of bovine growth hormone

In the preceding paper [Havel, H. A., Kauffman, E. W., Plaisted, S. M., & Brems, D. N. (1986) Biochemistry (preceding paper in this issue)], an associated intermediate was shown to be highly populated during the equilibrium denaturation of bovine growth hormone. In this paper, we describe its partial characterization and propose a mechanism for association. The associated equilibrium intermediate is populated under conditions that induce partial denaturation and at protein concentrations greater than 0.2 mg/mL. The remaining nativelike helical structure present in the partially denatured species is implicated in the mechanism of association as demonstrated by similar concentration dependencies and thermal stabilities of the helix and the associated equilibrium intermediate. Furthermore, it is suggested that a putative amphiphilic helix from residues 110-127 plays a critical role in the association as demonstrated by a diminution of the associated equilibrium intermediate when mixed with the peptide fragment 96-133. A model is proposed to account for these results in which partial denaturation exposes the segment of the protein corresponding to the hydrophobic face of the putative amphiphilic helix 110-127. This metastable form is the species from which association occurs. Association is stabilized by the hydrophobic interactions resulting from intermolecular packing of the lipophilic faces of the helices. The implications of these results to protein folding studies are described.     

Oxygenated cholesterols synergistically immobilize acyl chains and enhance protein helical structure in human erythrocyte membranes

Fourier transform infrared spectroscopy revealed that insertion of 20 alpha-hydroxycholesterol into human erythrocyte membranes (10% of total membrane sterol) immobilized the lipid acyl chains to a degree equivalent to enriching total membrane cholesterol by 50% (Rooney, M.W., Lange, Y. and Kauffman, J.W. (1984) J. Biol. Chem. 259, 8281-8285). Raman spectroscopy showed that the amount of acyl chain rotamers was not significantly altered by the presence of 20 alpha-hydroxycholesterol, indicating that acyl chain immobilization was limited to an inhibition of lateral motion. The presence of 20 alpha-hydroxycholesterol may synergistically enhance the acyl-chain-immobilizing behavior of membrane cholesterol. In addition, protein helical structure was not altered by 20 alpha-hydroxycholesterol. The insertion of 7 alpha-hydroxycholesterol into erythrocyte membranes resulted in an increase in protein helical structure which was comparable to that observed for erythrocyte membranes enriched with pure cholesterol by 50%. However, both acyl chain mobility and conformation were unchanged. These results suggest a synergistic behavior between oxysterols and cholesterol in modifying erythrocyte membrane packing.     

Salicylic acid blocks indomethacin-induced cyclooxygenase inhibition and lesion formation in rat gastric mucosa

Salicylic acid has been shown to decrease gastric mucosal lesions induced by indomethacin in the rat. In vitro, it has also been shown to counteract the inhibitory effect of indomethacin and aspirin on the cyclooxygenase enzyme system in seminal vesicle microsomes and in platelets and vascular tissue. The hypothesis that the mechanism of salicylic acid "protection" against indomethacin-induced gastric lesions involves interference with indomethacin-induced mucosal cyclooxygenase inhibition was tested. Male, fasted rats were treated with intragastric salicylic acid in doses of 50, 100, 200, 300, or 400 mg/kg concomitantly with a sc injection of 20 mg/kg of indomethacin. Gastric mucosal lesions and mucosal cyclooxygenase activity (as measured by ex vivo prostaglandin F2 alpha synthesis) were examined 3 hr later. Intragastric salicylic acid, 200-400 mg/kg, significantly reduced indomethacin-induced lesion formation, while counteracting significantly indomethacin inhibition of prostaglandin synthesis. Salicylic acid alone did not significantly change cyclooxygenase activity. It is concluded that topical salicylic acid can decrease indomethacin-induced gastric mucosal lesion in the rat, in part, by counteracting the inhibitory effect of indomethacin at the cyclooxygenase level.     

Raman spectroscopic and X-ray diffraction studies of the effect of temperature and Ca2+ on phosphatidylethanolamine dispersions

Raman spectroscopy and X-ray diffraction are used to study the effect of heat and Ca²⁺ on dimyristoylphosphatidylethanolamine dispersions. Unlike phosphatidylcholine dispersions, dimyristoylphosphatidylethanolamine bilayers (at pH 8) require heating above T_m in order for hydration to occur and apparently bind Ca²⁺ at very low levels. These results are related to models for membrane fusion.     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.