In vitro development of the hamster and chick secondary palate

A series of experiments were undertaken to compare the in vitro behaviour of the medial edge epithelium (MEE) of hamster, in which palatal shelves normally fuse, and chick, in which they do not fuse. Homotypic pairs of hamster and chick embryo palatal processes, single palatal processes, and heterotypic palatal shelves of both animals were grown in vitro. The results indicated that contact between palatal shelves may not be crucial for MEE differentiation in mammals. The ability to acquire pre-fusion characteristics may be present in mammalian palatal tissue from their early development and may be expressed by cessation of DNA synthesis in the MEE, elevation of cAMP, and MEE cell death. Isolated chick palatal shelf cultured under identical conditions did not express these mammalian pre-fusion characteristics. When MEE of hamster and chick palatal shelves were placed in contact with one another, the intervening epithelia underwent cytolysis. This could be due to either the destruction of chick MEE by lysosomal enzymes liberated from adjacent degenerating hamster MEE cells, or by induction of cell death in chick MEE by hamster mesenchyme. Heterotypic palatal tissue combinations also suggest that release of lysosomal enzymes in the hamster MEE, which leads to its dissolution, may be the terminal event in epithelial differentiation prior to the establishment of mesenchymal continuity. It is suggested that an inverse relationship exists between DNA synthesis and cAMP levels during palatogenesis: when palate closes (as in mammals) the MEE is eliminated by increasing cAMP levels, whereas when palate remains open (as in birds) low level of cAMP preserve the integrity of MEE by supporting DNA synthesis.     

Abortion due to infection with Chlamydia psittaci in a sheep farmer's wife.

A farmer''s wife who had helped with lambing aborted spontaneously in March after a short febrile illness in the 28th week of her pregnancy. She developed disseminated intravascular coagulation post partum with acute renal failure and pulmonary oedema. Recovery was complete after two weeks of hospital care. A strain of Chlamydia psittaci, probably of ovine origin, was isolated from the placenta and fetus. The patient''s serum showed rising titres of antibody against chlamydia group antigen; the placental and fetal isolates; and a known ovine abortion, but not a known avian, strain of C psittaci. IgG against both ovine abortion and enteric strains of C psittaci was detected, but IgM against only an abortion strain was detected. Histological examination showed pronounced intervillus placentitis with chlamydial inclusions in the trophoblast but no evidence of fetal infection or amnionitis. Laboratory evidence of chlamydial infection was found in an aborting ewe on the farm in January and in remaining sheep and lambs in July. Doctors should recognise the possible risk to pregnant women in rural areas where chlamydial infections in farm animals are widespread.     

Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

The respiratory burst of human neutrophils treated with various stimulators in vitro is dampened by exogenous unsaturated fatty acids

Cis-unsaturated free fatty acids (FFA) at concentrations between 10 and 30 microM suppressed the superoxide respiratory burst induced in human neutrophils by the chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine (FMLP). Corresponding trans-isomers had a reduced efficacy while saturated FFA were inert. The effects of unsaturated FFA were maximally achieved after several min of preincubation with cells and reversed upon washing. Increased concentrations of Ca2+ in the medium also relieved the inhibition. Unsaturated FFA were equally effective in dampening the respiratory burst induced by fluoride ions but less so with bursts elicited by 9 nM phorbol myristate acetate (PMA). Moreover reactions triggered by higher concentrations (e.g., 100 nM) of PMA were resistant to the effects of FFA. Radioimmunoassays showed that unsaturated FFA directly elevated intracellular cyclic adenosine monophosphate (cAMP) by severalfold above basal levels. It is suggested that inhibition is brought about by unsaturated FFA perturbation of the neutrophil membrane structure, perhaps with an independent contribution from a cAMP-dependent mechanism.     

An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

Fluorescent inhibitors of a cell surface protease used to locate leukaemia cells in kidney sections

Guanidinobenzoatase is a trypsin-like protease on the surface of cells capable of migration, for example leukaemia cells. We have used a number of fluorescent probes that are competitive inhibitors of guanidinobenzoatase to locate leukaemia cells in resin sections of kidney tissue obtained from leukaemic rats. We have demonstrated how this competitive inhibition system can be used to direct desired molecules (such as cytotoxic drugs) to these cells and to monitor the arrival of such compounds at the active site of guanidinobenzoatase. The principles developed in this study could equally well be applied to other enzymes on other cells provided suitable competitive inhibitors were designed. The presence of an enzyme on the surface of a cell can be used to direct molecules to that cell provided that these molecules contain a functional group that acts as an inhibitor for the chosen enzyme.     

Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.